!!EXPLORING THE MOLECULAR EVOLUTION OF PROTEINS WITH DEEP MUTATIONAL SCANNING By Matthew Steven Faber A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Biochemistry and Molecular Biology Ð Doctor of Philosophy 2019 !!ABSTRACT EXPLORING THE MOLECULAR EVOLUTION OF PROTEINS WITH DEEP MUTATIONAL SCANNING By Matthew Steven Faber In this thesis, deep mutational scanning is expanded and applied to better understand the molecular evolution of proteins. Protein evolution is a complex process where subtle changes in molecular architecture can have massive impacts on biophysica l properties, altering how well-adapted a protein is to a specific task or environment. Deep mutational scanning provides a finer level of understanding of molecular evolution by assessing the effect of every possible single -mutation on a protein Õs function. The technique combines site saturation mutant libraries, high throughput selections, and deep sequencing to tabulate the changes in mutant frequencies. From these changes the impacts of the mutations on protein function are characterized. This technology allows for efficient exploration of the local evolutionary landscape of a protein, making it a powerful tool for understanding evolution. Here, I use deep mutational scanning to study how the initial likelihood of obtaining the native folded state of an enzyme in vivo constrains its evolution. We designed two unique single -point mutants of AmiE, an aliphatic amidase from Pseudomonas aeruginosa . These mutant enzymes are significantly less likely to reach the native folded state in vivo than the unmutated precursor and have catalytic efficiencies that are statistically indistinguishable from the initial unmutated enzyme. I tested the impacts of nearly all single -point mutations for the two impaired enzymes using high -throughput growth selections and compared them to the precursor enzyme. These comparisons provided insights into how evolutionary outcomes are changed following !!decreases in the likelihood of native folding, and on how the impacts of single mutations combine to influence function. The other primary goal of this thesis is the development of a new method that expands the utility of deep mutational scanning studies. This method assembles comprehensive single -site saturation, and large multi -point, mutant genome libraries of the bacteri ophage !X174. To assemble the mutant genome libraries we combine nicking scanning mutagenesis and Golden Gate cloning. With these viral genome libraries, deep mutational scanning experiments can be performed in situ . These libraries are a valuable tool for studying the molecular determinants of viral host switching, the combination of inter - and intra -subunit mutations, and other aspects of the molecular evolution of viruses. !!iv This thesis is dedicated to my family and friends who have inspired and encouraged me to pursue a life of exploration through science. !!v ACKNOWLEDGEMENTS I want to acknowledge my graduate advisor, Tim Whitehead, for his guidance, suppo rt, and patience in helping me mature my scientific practices. Our time working together has significantly impacted my character in many positive ways and imbued me with the skills necessary to succeed. I also want to acknowledge my labmates - Angelica Medina -Cucurella, Carolyn Haarmeyer, Caitlin Stein, Emily Wrenbeck, James Stapleton, Justin Klesmith, Matilda Newton, Matt Bedewitz, Mon ica Kirby, and PJ Steiner - for their support and friendship. I want to thank my parents and siblings for their unending love, support , and for always encouraging me to be a curious and creative individual. I want to thank my friends for the great tim es spent together, and for the good times still to be had . Finally, I want to thank my wife Fabiola for her constant love , positivit y, support , and inspiration. !!vi TABLE OF CONTENTS LIST OF TABLES ÉÉÉÉÉÉÉÉ..ÉÉÉÉÉÉÉÉ...ÉÉÉÉÉÉÉ...ÉÉÉÉ. viii LIST OF FIGURES ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.ÉÉÉÉÉ.É..ÉÉ. x KEY TO ABBREVIATIONS É.ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.É.ÉÉÉÉ.É..É. xii CHAPTER 1 An introduction to deep mutational scanning, its limitations , and products ...1 Abstract ...ÉÉ ÉÉÉÉÉÉÉÉÉ.ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.. 2 Introduction ÉÉ É..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.. 3 The technology É É..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..ÉÉÉÉÉÉÉ... 3 Library preparation ÉÉÉÉÉÉÉÉÉÉÉÉ..ÉÉÉÉÉÉÉÉÉÉÉ.. 4 High throughput selections ÉÉÉÉÉÉ.ÉÉÉÉÉÉ.ÉÉÉÉÉÉÉÉ.. 5 The limitations ÉÉÉ ÉÉÉÉÉÉÉ....ÉÉÉÉÉÉ.ÉÉÉÉÉÉÉÉÉÉ..É 6 Experimental ÉÉÉÉÉÉÉÉ...ÉÉÉÉÉÉ.ÉÉÉÉÉÉÉÉÉÉ..É 6 Evolutionary insights ÉÉÉÉÉÉÉÉÉ..ÉÉ.ÉÉÉÉÉÉÉÉÉÉ..É 7 The product s ÉÉÉÉ ÉÉÉÉÉÉÉ....ÉÉÉÉÉÉ.ÉÉÉÉÉÉÉÉÉÉ..É 9 Growth selection products ÉÉÉÉÉÉ..ÉÉÉ.ÉÉÉÉÉÉÉÉÉÉ..É 9 YSD-FACS products ÉÉÉÉÉÉÉÉÉÉÉ..ÉÉÉÉÉÉÉÉÉÉ.É 12 REFERENCES ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ 14 CHAPTER 2 !Data -driven engineering of protein therapeutics ÉÉ.ÉÉÉ...ÉÉ.ÉÉÉ 18 Abstract ...ÉÉ ÉÉÉÉÉÉÉÉÉ.ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ 19 Introduction ÉÉÉ..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ ÉÉÉÉÉÉÉ 20 Large -scale mutational anal ysis ÉÉÉÉÉÉ..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ 22 Antibody deep mutational scan ning É...ÉÉÉ..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ 22 Enzyme deep mutati onal scanni ng ..É...ÉÉÉ..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ 24 Writing libraries of synthetic genes É...ÉÉÉ..ÉÉÉÉÉÉÉÉÉÉÉ ÉÉÉÉ 25 Nature -sourced data É ...ÉÉÉ..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ 27 Protein design by phylog eny É...ÉÉÉ..ÉÉÉÉÉÉÉ..ÉÉÉÉÉÉ .É 27 Engineering from human antibo dy repertoires ÉÉÉ ÉÉÉÉÉÉÉÉÉ.... 28 Perspective É...ÉÉ É..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..ÉÉÉÉÉ 29 Acknowledgements ÉÉÉÉÉÉÉÉÉÉ É..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ 30 REFERENCES ....ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ 31 CHAPTER 3 Impact of in vivo protein folding probability on local fitness landscapes .É 37 Abstract ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ...É. É 38 Introduction É ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.. 39 Results ÉÉÉ ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.. 42 AmiE variants with lower in vivo folding probabilities and wild -type catalytic efficiencies engineered ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ .ÉÉÉÉÉÉÉÉÉ 43 Deep mutational scans for AmiE variants ÉÉÉÉÉÉÉÉÉÉÉÉÉÉ ... 47 !!vii Distribution of beneficial fitness effects are largely insensitive to initial in vivo protein folding probability ÉÉÉÉÉÉÉÉ.ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.. 49 Moderate epistasis observed with decreasing enzyme in vivo folding probability ... .......................ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.ÉÉ...É... 51 Most beneficial mutations in the WT background are shared ..ÉÉÉÉÉ..É. 52 Positive sign epistasis is o verwhelmingly specific ÉÉÉÉÉÉÉÉÉÉ..É 55 A plurality of unique beneficial mutations is codon -dependent ÉÉÉÉÉ..É 55 Discussion É ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ...É. 57 Materials and m ethods É ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..ÉÉÉ..ÉÉ. 59 Reagents ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ .ÉÉÉÉ..ÉÉ 59 Computational design of folding impaired mutants ÉÉÉÉÉ ÉÉÉÉ.ÉÉ 59 Plasmid construction ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ ÉÉÉ 60 Near comprehensive single -site mutant l ibrary construction É ÉÉÉÉÉÉ... 61 Protein expression and pu rification ÉÉ ÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..... 61 Biophysical analysis o f proteins ÉÉ ÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..ÉÉ 62 Growth selecti ons ÉÉÉÉÉ .ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..É.. 65 Deep mutational s canning É .ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..É. 66 Data availabi lity ÉÉÉ.ÉÉ...ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..ÉÉÉ 67 Acknowledgemen ts ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ .É.ÉÉ 68 REFERENCES ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.É.É.. 69 CHAPTER 4 Saturation mutagenesis genome engineering of infective !X174 bacteriophage via unamplified oli go pools and golden gate assembly É....ÉÉÉÉÉÉ.74 Abstract ... ..ÉÉÉÉÉÉÉÉÉÉÉ.ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..ÉÉ 75 Introduction ÉÉ É..É..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..ÉÉ 76 Results ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.. ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ 77 Discussion ÉÉÉ. .ÉÉÉÉÉÉÉÉÉÉÉÉÉÉ....ÉÉÉÉÉÉÉÉÉÉÉ.. 81 Materials and meth ods ÉÉÉÉÉ ÉÉÉÉÉÉÉÉÉÉ..ÉÉÉÉÉÉÉÉÉ... 83 Reagents ÉÉÉÉÉÉÉÉÉÉÉÉ..ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ. 83 Segmentation of the "X174 genome ÉÉÉÉÉÉÉÉÉÉÉ ..ÉÉÉÉÉ. 83 Introduction of nicking site ÉÉÉÉÉ ÉÉÉÉÉÉÉÉÉÉÉ..ÉÉÉ.... 83 Comprehensive single site mutant lib rary construction ÉÉÉÉÉÉÉÉ ..É. 84 Illumina sequencing prep and analysis ÉÉÉÉÉÉÉÉÉÉÉ ..ÉÉÉ..É 85 Assembly of mutant genomes ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ ....É.. 85 REFERENCES ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ.ÉÉÉÉÉÉÉ...É 87 CHAPTER 5 Conclusions and perspectives ÉÉÉÉÉÉ...ÉÉÉÉÉ...ÉÉÉÉÉ...... 91 REFERENCES ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ..ÉÉÉÉÉÉ..ÉÉ 95 APPENDICES ÉÉ.ÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉÉ... 97 APPENDIX A Chapter 3 sup porting information ÉÉÉ ...ÉÉ..ÉÉÉ..ÉÉ..ÉÉ.. 98 APPENDIX B Chapter 4 supporti ng information ÉÉÉ ...ÉÉÉÉÉÉÉÉ..ÉÉ 145 APPENDIX C Purification of the TROP2 extracellular domain from a stable insect cell line using ammonium sulfate precipitation ÉÉÉÉ..ÉÉÉ..ÉÉÉÉÉ.ÉÉÉÉÉ...É 161 REFERENCES......... .......................................................................................................179 !!viii LIST OF TABLES Table 4.1: Mutant library NGS statistics .......................................................................................80 !Table 4.2: Mutant genome assembly statistics ..............................................................................81 Table A 1: Relative fitness for I38V and I22L synonymous codons in the AmiE WT background . ......................................................................................................................................................122 Table A 2: Biophysical analysis of the AmiE variants ................................................................123 Table A 3: Thermal shift analysis data and statistics ...................................................................124 Table A 4: Circular dichroism analysis of thermal denaturation statistics ..................................125 Table A 5: DNA sequences of transcriptional elements in pl asmids used for deep mutational scans .............................................................................................................................................126 !Table A 6: Summary of MG1655 rph+ transformants obtained during selection strain mutant library preparation ........................................................................................................................127 Table A 7: Summary of mutational library statistics ...................................................................128 Table A 8: Goodness -of-fit test statistics for distribution fittings ...............................................129 Table A 9: Deleterious empirical cumulative distribution function (ECDF) analysis statistics ..130 Table A 10: Inner and outer primers for PCR reactions for Illumina sequencing .......................131 Table A 11: Beneficial mutations shared by all enzymes ............................................................133 Table A 12: Beneficial mutations shared by only AmiE WT and AmiE I122L ..........................136 Table A 13: Beneficial mutations shared by only AmiE WT and AmiE I38V ...........................137 Table A 14: Beneficial mutations shared by only AmiE I38V and AmiE I122L ........................138 Table A 15: AmiE WT unique beneficial mutations ...................................................................139 Table A 16: AmiE I122L unique beneficial mutations ................................................................140 Table A 17: AmiE I38V unique beneficial mutations .................................................................141 !!ix Table A 18: AmiE WT unique beneficial mutations with synonymous codon fitness disparities .... ......................................................................................................................................................142 Table A 19: AmiE I122L unique beneficial mutations with synonymous codon fitness disparities . ......................................................................................................................................................143 Table A 20: AmiE I38V unique beneficial mutations with synonymous codon fitness disparities .. ......................................................................................................................................................144 !Table B 1: Mutant library preparation summary ....................................................................158 Table B 2: Primers for incorporating BbvCI nicking sites into the pCR2.1 -topo shuttle vector ....... ......................................................................................................................................................159 !Table B 3: Inner and outer primers for PCR reactions for Illumina sequencing ........................160! !!x LIST OF FIGURES Figure 1.1: Deep mutational scanning overview .............................................................................4 Figure 2.1: High throughput techniques used to overcome therapeutic protein engineering bottlenecks .....................................................................................................................................21 Figure 2.2: Big data yields insights for efficient engineering of protein therapeutics ...................29 Figure 3.1: Design of deep mutational scanning experiment ........................................................44 Figure 3.2: Local fitness landscapes are nearly insensitive to initial protein folding probability in vivo .................................................................................................................................................50 Figure 3.3: Positive sign epistatic mutatio ns are spatially segregated and specific .......................54 Figure 3.4: Unique beneficial mutations have high percentages of synonymous codon fitness disparities .......................................................................................................................................56 !Figure 4.1: "X174 mutant library assembly .................................................................................79 !Figure A 1: Chromatogram of purified AmiE variants ................................................................102 !Figure A 2: Representative far -UV spectra of the folded and unfolded AmiE variants ..............103 !Figure A 3: Locations of I38V and I122L mutations in AmiE quaternary structure ...................104 !Figure A 4: AmiE WT tryptophan emission spectra as a function of GDN -HCl concentrati on..105 !Figure A 5: Thermal shift analysis of the purified AmiE variants ..............................................106 !Figure A 6: Activity loss of AmiE WT following dilution ..........................................................107 !Figure A 7: Thermal denaturation monitored by far -UV circular dichroism ..............................108 !Figure A 8: Frequency distribution of pre-selection read counts for AmiE libraries ..................109 !Figure A 9: Deep mutational scanning replicates for AmiE WT compared with previous literature .......................................................................................................................................110 !Figure A 10: Relative fitness metrics for AmiE proteins as a function of pre -selection read counts ......................................................................................................................................................111 !!!xi Figure A 11: Normalized distribution of fitness effects for WT, I122L, and I38V backgrounds ..... ......................................................................................................................................................112 Figure A 12: Analysis of proportions of deleterious mutations ...................................................113 Figure A 13: Venn diagram of shared and unique beneficial mutations using the strict cutoff ..114 !Figure A 14: Correlation analysis of linear regression normalized shared beneficial mutations .115 !Figure A 15: Dot plots of fitness effect synonymous codon variances for beneficial mutat ions ....... ......................................................................................................................................................116 !Figure A 16: SDS -PAGE analysis of the purity of the purified AmiE variants ..........................117 !Figure A 17: Comparison of AmiE I122L outlier mutation technical replicates ........................118 !Figure A 18: AmiE WT heatmap .................................................................................................119 !Figure A 19: AmiE I122L heatmap .............................................................................................120 !Figure A 20: AmiE I38V heatmap ...............................................................................................121 !Figure B 1: Introduction of nicking sites into shuttle vectors conta ining viral genes .................149 !Figure B 2: F1 heatmap of counts ................................................................................................150 !Figure B 3: F2 tile 1 heatmap of counts .......................................................................................151 !Figure B 4: F2 tile 2 heatmap of counts .......................................................................................152 !Figure B 5: F3 tile 1 heatmap of counts .......................................................................................153 !Figure B 6: F3 tile 2 heatmap of counts .......................................................................................154 !Figure B 7: G1 tile 1 heatmap of counts ......................................................................................155 !Figure B 8: G1 tile 2 heatmap of counts ......................................................................................156 Figure B 9: G2 heatmap of counts ...............................................................................................157 Figure C 1: TROP2Ex and its expression ....................................................................................165 Figure C 2: Purification of TROP2Ex from insect cell cultures ..................................................167 Figure C 3: Verification of the proper assembly of the m7E6 scFv yeast display construct and of binding to TROP2Ex ...................................................................................................................168 !!xii KEY TO ABBREVIATIONS AmiE, Amidase E CDR, complementarity determining regions DFE, distribution of fitness effects DMS, deep mutational scanning FACS, fluorescence activated cell sorting FPLC-SEC, fast protein liquid chromatography with a size exclusion chromatography gel IMAC, immobilized metal affinity chromatography KS, Kolmogorov -Smirnoff NSM, nicking scanning mutagenesis scFv, single chain fragment of variable regions VH-VL, variable heavy and variable light chains TROP2Ex, t umor associated calcium signal transducer 2 extracellular domain YSD, yeast surface display !! 1 CHAPTER 1 An introduction to deep mutational scanning, its limitations, and products ! 2 Abstract Deep mutational scanning combines saturation mutagenesis, high throughput selections, and deep sequencing to characterize the impacts of individual mutations on a respective protein. This technology allows for the characterization of thousands of mutations in parallel and as such has greatly expanded our abilities to understand protein evolution and perform rational design. This chapter wil l provide an introduction to deep mutational scanning, its methodological limitations, and what the outputs can and cannot tell us about the impacts of the tested mutations. ! 3 Introduction Deep mutational scanning (DMS) is a platform technology for efficiently assessing the impact of thousands of individual mutations on a protein of interest 1,2 (Figure 1.1 ). A DMS experiment requires a high throughput selection (HTS) for the protein of interest, the ability to generate comprehensive si ngle -site saturation mutant libraries, and access to a deep sequencing platform. DMS is facilitated by clever cellular and molecular biology methods for HTS and for library preparations, as well as by the drop in deep sequencing costs to pennies per millio n base -pairs. The outputs of DMS are fitness metrics for each mutation that describe the magnitude by which a given mutation is beneficial or deleterious in the HTS performed. This chapter is not a comprehensive review of deep mutational scanning as there are already several excellent reviews that do so 3,4 . Additionally, this chapter will not focus on the application of the obtained datasets for studying evolution, or for forward engineering as these aspects have been reviewed in Wrenbeck et al. 5 and Faber and Whitehead 6 (Chapter 2). This chapter will provide a brief introduction to the technical aspects of DMS, what and where the limitations in the methods are, and what the obtained datasets can and cannot tell us about the mutations studied. The techn ology As mentioned above DMS is the combination of three different processes: preparation of comprehensive site -saturation mutant libraries, high throughput selections, and deep sequencing (Figure 1.1 ). Here I will briefly describe advances in methods for preparing the mutant libraries, and the HTSs most used in our laboratory. ! 4 Library preparation Single site saturation (SSM) mutagenesis libraries are constructed by template based mutagenesis using plasmid ssDNA and mixtures of mutagenic primers that encode the desired mutation(s). The mutant strand is ligated, the parent DNA strand digested, and a second !"#$%& '()*()+& ,-) &./01,"$2")%& 3,14"$& 5-3,"$$(1)& %#+& 5)678"& 9,1:%;& <04$%,#%"& !"#$%&$0,=#2"&*($3/#7& 9,1:%;&$"/"2%(1)& DITTYXITTYDXTTYDIXTYDITXYDITTXX$04$%(%0%"*&%1&#//& #8()1(*$&#)*& #&$%13&21*1)& !">#,(#)%& ?10)%$&& @3,"A$"/"2%(1)B& ?10)%$&& @31$%A$"/"2%(1)B& #"#"#"#"#"$%"&'"#"("&"<"/"2%& %#,+"%& ?1)$%,02%& /(4,#,7& C(+;& %;,10+;30%& $"/"2%(1)& D""3&$"E0")2()+& F1,8#/(6"*& =(%)"$$&8"%,(2& 100101102PEWSTOP *FWYPSTART MILVAGCSTNQDEHKRHydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged '")"=(2(#/& D"/"%",(10$& F"0%,#/& Figure 1.1: D eep mutational scanning overview . A DMS experiment begins with target selection, the selected target has to be compatible with a HTS. Next, comprehensive site -saturation mutant libraries are created. The HTS is applied to the mutant libraries to cause the weaker mutants to drop out, and beneficial mutations to proliferate. Counts of mutants in the pre - and post -selection libraries are tabulated using deep s equencing , and normalized fitness metrics are calculated based on the performance of the unmutated protein . ! 5 replication and ligation is performed 7. Finally, the library is transformed into a cloning strain of E. coli to generate enough plasmid library for downstream applications. Initial library preparations using the Pfunkel method required preparation of ssDNA from bacteriophage and used uracil -containing DNA, which increased time and effort 8. During my PhD studies my lab invented Nicking Mutagenesis, which allowed for the production of ssDNA by nicking a single strand of a dsDNA plasmid and digesting i t with exonucleases 8. This key advance allowed SSM library preparation from plasmid dsDNA in a single day. We have further advanced the production of these mutant libraries by replacing the degenerate mutagenic oligos with unamplified ink -jet printed oligo pools that contain user -defined mutations 9 (Chapter 5). Using unamplified oligo pools in place of degenerate oligos both simplifies the mutagenesis procedure and results in libraries with greater representations of all of the programmed mutations 9. High throughput selections The type of HTS used in a DMS experiment is dependent on the protein(s) being studied and the information one wants to obtain 5,10 . The two primary screens used in our laboratory are yeast surface display (YSD) paired with fluorescen ce activated cell sorting (FACS) 9, and growth -based selections Ð where weaker variants become depleted and stronger variants enriched - for studying enzymes 10-12 (Chapter 3; Figure 1.1 ). Other selections exist, such as phage display in place of yeast displ ay, lytic virus growth selections, or reporter based assays for enzyme function. Pairing YSD and FACS can be used to study protein -protein interactions, improve/engineer affinity and specificity 13-16, and also to map paratopes and epitopes 17-18. YSD and FA CS can also be used to improve/study the stability of proteins and enzymes 19-21. DMS experiments with growth -based selections can be used to inform the affinity/specificity ! 6 engineering of enzymes, and to improve enzymes for a respective reaction environmen t12 (Chapter 3). Both YSD -FACS, and growth -based selections, can be used to answer molecular evolution questions because these methods map local fitness landscapes for a given environmental condition 12,20 (Chapter 3). Thus, DMS is a valuable tool for answe ring both applied and fundamental research questions. The limitations Experimental DMS experiments require a suitable HTS, which significantly restricts the reach of these techniques. For YSD paired with FACS the desired target for scanning must be ab le to be displayed on the yeast cell surface, and the purified binding partners must be able to be produced 10,17,18 . Additionally, the reaction conditions have to be compatible with FACS and non -toxic to the displaying cells. Altered glycosylation pattern s Ð inherent in YSD Ð can also impose significant issues. The mannose rich O - and N -linked glycosylation patterns in yeast are distinct from those in mammals, and cytosolic proteins are naturally unglycosylated. The attachment of non-native glycosylations can disrupt binding and alter other biophysical parameters for a protein. Also restricting the use of FACS is the oligomeric state of the protein being displayed and our group has successfully displayed up to homotrimers on surface of yeast 17. Growth -based HTSs require that cell growth be dependent on the reaction catalyzed by the enzyme of study. In Chapter 3 we study an amidase using DMS. When supplied with an amide the amidase releases ammonium, allowing for nitrogen restricted growth selections. Grow th selections also require a growth rate ratio Ð growth rate in selective media over growth rate in unselective media Ð that is within a window of values 10. Outside of the window the ! 7 growth of the cell is not proportional to the function of the enzyme. Tun ing the growth rate ratio to be within this window requires certain biophysical parameters of enzymes like solubility, and catalytic efficiency to be met, as well as transcription and translational tuning with synthetic elements. Therefore, enzymes with ve ry low catalytic efficiencies, and/or low solubilities, are unlikely to be compatible with DMS. DMS is subject to the limits of the current mutant library preparation methods, cell sorting restrictions, and limitations in sequencing technologies 9,10,18,2 2. The information obtained from DMS experiments is also restricted to a range of fitness metrics. DMS experiments performed using the pipelines developed in our lab can only scan an ~5 -fold range of differences in fitness metrics 20,23 . DMS can discriminat e sign differences with great accuracy and fidelity, but the ability to accurately quantify the impact of very deleterious mutations is dependent on sequencing depth 10-12 (Chapter 3). Often very deleterious mutations can only be qualitatively described. When analyzing obtained datasets we are limited in the conclusion we can draw about the biophysical impacts of mutations. This limitation is typical in HTSs as these screens are often general competition assays that do not discriminate between the various biophysical properties that can be modified. Additional HTS screens 19,20 that select for a specific biophysical property, or experimental characterization of individual mutants 12 are required to determine how mutations impact the biophysical properties of the protein. Evolutionary insights DMS datasets have to be approached with the understanding that these are limited test tube evolution experiments. Additionally, in any selection you get what you select for, meaning ! 8 that the conclusions and insights we obtain are biased for, and limited to, the selections performed 10,12,20 (Chapter 3). In Chapter 3, we perform our selections in a highly controlled environment at 37¡C. However, our model enzyme - Amidase E from P. aeruginosa - has naturally evolved to function in a wide range of temperatures 24. It is likely that mutations that are beneficial or deleterious at 37¡C could have drastically different outcomes at different temperatures 25. Additionally, our model enzyme is promiscu ous and able to digest many short chain amides 12,24 , yet the selections we perform in Chapter 3 only use one substrate, acetamide. Therefore, we have selected for mutants that are only better at catalyzing the digestion of acetamide, or that increase the a mount active enzyme in vivo at 37¡C in the specific cellular host. It is possibly that the beneficial mutations found in Chapter 3 will have decreased catalytic efficiencies with other substrates, and that some of the deleterious mutations found might be beneficial for digesting other aliphatic amides 12 (Chapter 3). Selection conditions inherently constrain and bias the outcomes of test tube evolution experiments 11,12,26,27 . In laboratory evolution experiments the use of a single set of environmental cond itions Ð which produce fitness values that can accurately describe mutational impacts only in the respective environment Ð is often required to limit the complexity of evolution such that it can be studied and understood 27. For example, the Lenski evolutio n experiments have studied molecular and organismal evolution in several E. coli strains, individually, at a single temperature and constant chemical environment for over 60,000 generations 28,29 . The evolutionary insights obtained from this experiment are extremely valuable, yet they are biased for the organisms studied and the selection conditions used. By comparison, DMS approaches to studying molecular evolution are also subject to these limitations and biases. The environment used in a DMS experiment is user defined within certain parameters, and the selection ! 9 environment can be modified as long as the HTS is maintained 10. However, altering the conditions of the HTS to test other environments Ð for example by performing growth -based selections at severa l different temperatures Ð can be extremely challenging and may not be feasible. Finally, DMS is currently unable to model evolution over thousands of years and in highly complex environments. In nature there are many different environments a single orga nism can pass through, and many other organisms and pathogens to compete with, making the process of evolution massively more complex than in our laboratory experiments 30-33. Therefore, while the data obtained from DMS is very informative, we must never lo se sight of the fact that what is true in the test tube is an oversimplification of what is possibly occurring in nature. The products Deep mutational scans provide local fitness landscapes typically defined as nearly all single mutational steps in th e evolutionary space 1,2 (Figure 1.1 ). These can be used to gain insights into molecular evolution 12,20 (Chapter 3) and can be used in forward engineering proteins toward a desired purpose 5,6 (Chapter 2). As described previously, the definition of fitness i s dependent on the HTS used and the environment of the assay. The obtained normalized fitness metrics describe how well a respective mutation is able to compete with the unmutated predecessor protein in the respective HTS 10. Growth selection products With growth based selections for studying enzymes, the normalized fitness metric describes how well a cell hosting a respective mutant is able to compete with Ð replicate faster ! 10 than Ð a cell hosting the unmutated enzyme in the assaying conditions at a respe ctive temperature 10. The mathematics for calculating normalized fitness metrics for growth based selections Ð as in Chapter 3 - from deep sequencing data was published by Kowalsky et al. 10 and is as follows. First, the frequency of a respective mutant ( i) in the pre - (fio) and post -selection ( fif) populations is calculated with the tabulated counts of mutants from the deep sequencing data: !!"!!!!"!!" (1) !!"!!!!"!!" (2) Where xio and xif are the number of counts of a specific variant in the pre - and post -selection populations respectively, and !xoi and !xfi are the total number of counts for all variants in the pre- and post -selection populations respectively. Next, the enrichment ratio, !i, for a mutant in the population is calculated from frequencies of the mutants obtained in (1) and (2): !!!!"#!!!"!!" (3) The equation for calculating the enrichment ratios can be rewritten as: !!!!!"#!!!"!!"!!!!"#!!!!"!!"! (4) We also write the equation for the specific growth rate ( µi) (5) of a cell as: !!!!"!!"!!"!! (5) Where t is the difference in time between the end of the selection ( f) and initiation of the selection ( o). Comb ining the equations (4) and (5) gives: !!!"#!!!!!!!!!!!!!"#!!!"!!"! (6) ! 11 To simplify the equation we can first generate the average doubling period ( !!) as a function of the change in the total number of counts for all variants i n the pre - and post -selection populations: !!!!"#$%& !!"!!"#$%&'() !!!"#!!!!"!!"! (7) We can also remove t through redefining it: !!!!"!!!!!! (8) Where !! is the bulk average growth rate of the po pulation for the time between the initiation and end of the selection. Next we combine (7) and (8) into (6) to make the growth rate of a mutant a function of its respective enrichment ratio: !!!!!!!!!!!!!!! (9) Finally, we can calculate ou r normalized fitness metric ( !!) where the growth rate of the respective mutant protein (i) is normalized to that of the unmutated predecessor protein (wt): !!!!!"#!!!!!!"! (10) We can also set the normalized fitness metric as a functi on of the enrichment ratios and average doubling period: !!!!!"#!!!!!!!!!"!!!! (11) The normalized fitness metric provides a quantitative description of how well a cell harboring a mutant i is able to compete with a cell hosting the p redecessor enzyme in the respective environment. This data does not provide information on how the mutation impacts the biophysics of the enzyme. ! 12 For enzyme -based DMS, there are two general biophysical factors that are impacted by mutations and that dete rmine fitness outcomes: specific velocity, and the amount of active enzyme being expressed within the cell. Both of these general properties are composed of a variety of different factors. Specific velocity is dependent on the standard Gibbs free energy of the reaction being catalyzed, substrate and product concentrations, flux of upstream and downstream reactions, the Michaelis constant (K M), and the maximum reaction velocity (kcat)25,34 . The probability of the enzyme being expressed and properly folded is a function of: the folding rate, the fidelity of the association process for oligomeric proteins, and the thermodynamic stability of the tertiary and quaternary structures and intermediates 35,36 . A growth selection is unable to discriminate between any of the above -mentioned factors, and additional biophysical analysis is required to understand why a mutation is deleterious or beneficial. YSD -FACS products This chapter will not present the mathematics used in calculating the normalized fitness metrics for DMS experiments with YSD -FACS, those calculations can be found in the referenced work 10,17 . While I have performed DMS with YSD -FACS for epitope mapping 17, paratope mapping (results incorporated into a patent in preparation), and affinity maturation (results incorporated into a patent in preparation), these projects are not included in this thesis. Normalized fitness metrics obtained from YSD -FACS experiments provide a quantitative measure of how well a mutant protein competes with its unmutated ancest or in either binding to a given target, or in displaying on the surface of the yeast in a respective environment 10,17,20 . Unlike the growth -based selections, YSD -FACS experiments can select for two different general biophysical properties: affinity/specifi city, and surface display ( Figure 1.1 ). ! 13 Comparing the obtained fitness landscapes from selections for binding/display with those from selections only for display allows for the discrimination of mutations that impact stability/solubility from those that impact affinity/specificity 17,19,20 . Selections for display can be used to select for more stable variants, and to allow us to identify mutations that alter folding probabilities. To aid in discriminating the impacts of the mutations Kowalsky et al. 17 calculated Shannon entropy metrics for each position in the protein being studied for both the displaying sorted and binding/display sorted populations. These Shannon entropy metrics provide a quantitative measure of how well the respective position tolerates mutations. Positions with the lowest Shannon entropy scores are the least able to tolerate mutations. Comparison of Shannon entropy scores from selections for display with selections for binding/display allows for the identification of residues critical fo r the binding reaction. This comparative analysis is the foundation for mapping protein -protein interactions 17,18 with DMS. As described in Chapter 2, comparison of DMS datasets from different HTS with the same target protein allows for greater discriminat ion of the biophysical implications for respective mutations, and how this data can be used for forward engineering. ! 14 REFERENCES ! 15 REFERENCES "#!Fowler DM, Araya CL, Fleishman SJ, Kellogg EH, Stephany JJ, Baker D, Fields S: High -resolution mapping of protein sequence Ðfunction relationships . Nat Methods 2010, 7:741 - 746. !$#!Hietpas RT, Jensen JD, Bolon DNA: Experimental illumination of a fitness landscape . Proc Natl Acad Sci U S A 2011, 108:7896 -7901. !%#!Araya CL, Fowler DM (2011) Deep mutational scanning: Assessing protein function on a massive scale . Trends Biotechnol 29(9):435 Ð442. !&#!Fowler DM, Fields S: Deep mutational scanning: a new style of protein science . Nat Methods 2014, 11:801 Ð807. !'#!Wrenbeck EE, Faber MS, W hitehead TA: Deep sequencing methods for protein engineering and design . Curr Opin Struct Biol 2017, 45:36 -44.Wrenbeck 2019 !(# Faber MS, Whitehead TA: Data -driven engineering of protein therapeutics . Curr Opin in Biotech 2019, 60:104 -110. !)# Firnberg E, Ostermeier M : PFunkel: efficient, expansive, user - defined mutagenesis . PLoS ONE 2012, 7:e52031. !*#!Wrenbeck EE, Klesmith JR, Stapleton JA, Adeniran A, Tyo KEJ, Whitehead TA: Plasmid -based one -pot saturation mutagenesis . Nat Methods 2016, 13:928-930. !+#!!"#$%& ,!"#"$%&&'()*+(,-%./%$(01+(2'3%$(4,+(5%&-$6/(1+(57$%&&.()+(8.$39(45+(!"-&%$(,:+( -./01.123!456! !"#$ %&#'()#&*"()+,#*-./*01/2+#)#"("*1"()+*1)-1$('(#&*.,(+.*-..," #!!"#$%&'()%&*+,%&-+.% !$7"+8!49:649:#! !!"7#!Kowalsky CA, Klesmith JR, Stapleto n JA, Kelly V, Reichkitzer N, Whitehead TA: High -Resolution Sequence -Function Mapping of Full -Length Proteins . PLoS One 2015, 10:e0118193. !""# Klesmith JR, Bacik JP, Michalczyk R, Whitehead TA: Comprehensive Sequence -Flux Mapping of a Levoglucosan Utilization Pathway in E. coli . ACS Synth Biol 2015, 4:1235 Ð1243. !"$#!Wrenbeck EE, Azouz LR, Whitehead TA: Single -mutation fitness landscapes for an enzyme on multiple substrates reveal specificity is globally encoded . Nat Comm 2017, 8:15695. !! 16 "%# White head TA, Chevalier A, Song Y, Dreyfus C, Fleishman SJ, Mattos CD, Myers CA, Kamisetty H, Blair P, Wilson IA, Baker D: Optimization of affinity, specificity and function of designed influenza inhibitors using deep sequencing . Nat Biotechnol 2012, 30:543 -548. !"&# Strauch E -M, Fleishman SJ, Baker D: Computational design of a pH -sensitive IgG binding protein . Proc Natl Acad Sci U S A 2014, 111:675 -680. !"'# Procko E, Berguig GY, Shen BW, Song Y, Frayo S, Convertine AJ, Margineantu D, Booth G, Correia BE, Cheng Y et al.: A computationally designed inhibitor of an Epstein -Barr viral Bcl -2 protein induces apoptosis in infected cells . Cell 2014, 157:1644 -1656. !"(# Koenig P, Lee CV, Walters BT, Janakiraman V, Stinson J, Patapoff TW, Fuh G: Mutational landscape of antibody variable domains reveals a switch modulating the interdomain conformational dynamics and antigen binding . Proc Natl Acad Sci U S A 2017, 114:E4 86-E495. !")#!Kowalsky CA, Faber MS, Nath A, Dann HE, Kelly VW, Liu L, Shanker P, Wagner EK, Maynard JA, Chan C et al.: Rapid fine conformational epitope mapping using comprehensive mutagenesis and deep sequencing . J Biol Chem 2015, 290:26457 -26470. !"*#!Medina -Cucurella AV, Whitehead TA: Characterizing Protein -Protein Interactions Using Deep Sequencing Coupled to Yeast Surface Display . Met in Mol Biol 2018, 1764:101 -121. !"+# Julian MC, Li L, Garde S, Wilen R, Tessier PM: Efficient affinity maturation of antibod y variable domains requires co -selection of compensatory mutations to maintain thermodynamic stability . Sci Rep 2017, 7:45259. !$7# Klesmith JR, Bacik JP, Wrenbeck EE, Michalczyk R, Whitehead TA: Trade -offs between enzyme fitness and solubility illu minated by deep mutational scanning . Proc Natl Acad Sci U S A 2017, 114:2265 -2270. !$"#!-;1<=1>?!@@8!9131A/0B!C58!DE1FG/0.!HI8!JKF./#)#/(:*&$2'/?*"#,#:/(4#*()/#$'#$#):#?*2)&*-.-1,2/(.)*+#)#/(:"*.'*$2-(&* 2&2-/2/(.)#!/((5&9+:&';#.&':#.&-1,$& $7"%8!&&6"+' ,$"'#!!!%$#!P/1=1;G2532G!5H8!T;/ 1=1!UT8!D/F.K#)#/(:* 42$(2/(.)*.'*2*<2:/#$(2,*-2/5.+#)*;(/5()*()&(4(&12,"*;(/5*:7"/(:*'(<$."("*-$.4(&#"*2* $#:.$&*.'*"#,#:/(4#*-$#""1$#" #!<=$5"+&>+(& $7"&8!&(6*$ ,*)#!!%%# Liao M, Somero GN, Dong Y: Comparing mutagenesis and simulations as tools for identifying functionally important sequence changes for protein thermal adaptation. Proc Natl Acad Sci U S A 2019, 116:679 -688. !%&#!Siddiqui KS: Defying the activity Ðstability trade -off in enzymes: taking advantage of entropy to enhance activity and thermost ability . Critical Reviews in Biotechnology 2017, 37:309 -322. !%'#!Baker D, Agard DA: Kinetics versus thermodynamics in protein folding . Biochemistry 1994, 33(24):7505 -7509. !%(# Shakhnovich EI: Theoretical studies of protein -folding thermodynamics and kinetics . Curr Opin in Struc Biol 1997, 7:29 -40. !!!!!!!! 18 CHAPTER 2 Data -driven engineering of protein therapeutics This chapter is adapted with permission from the article ÒData -driven engineering of protein therapeuticsÓ in Current Opinion in Biotechnology 60:104 Ð110 by Matthew S. Faber and Timothy A. Whitehead. Copyright 2019 Elsevier Ltd. ! 19 Abstract Protein therapeutics requires a series of properties beyond biochemical activity, including serum stability, low immunogenicity, and manufacturability. Mutations that improve one property often decrease one or more of the other essential requirements for therapeutic efficacy, making the protein engineering challenge difficult. The past decade has seen an explosion of new techniques cent ered around cheaply reading and writing DNA. This review highlights the recent use of such high throughput technologies for engineering protein therapeutics. Examples include the use of human antibody repertoire sequence data to pair antibody heavy and lig ht chains, comprehensive mutational analysis for engineering antibody specificity, and the use of ancestral and inter -species sequence data to engineer simultaneous improvements in enzyme catalytic efficiency and stability. We conclude with a perspective o n further ways to integrate mature protein engineering pipelines with the exponential increases in the volume of sequencing data expected in the forthcoming decade. ! 20 Introduction Over 100 therapeu tic proteins are currently FDA -approved for use as drugs, with functions as variegated as enzymes that rob tumors of necessary nutrients to antibodies that block signaling mechanisms important for clinical presentation of rheumatoid arthritis. Regardless of the exact category of protein, there are a large number of requirements necessary for therapeutic efficacy: the biologic must possess sufficient affinity or catalytic efficiency toward its intended target while minimizing non -specific interactions, harbor a sufficient in vivo half -life, and maintain low immunogenicity and aggregation propensity. Additionally, each protein must satisfy a number of manufacturability constraints such as the capability of expression and purification in sufficient quantities, sufficient storage stability, and so forth. Natura lly occurring proteins usually are suboptimal in at least one of these properties. While alterations to these properties can be imparted by mutations (protein engineering), mutations are pleiotropic and often a property can only be improved at the expense of another important requirement. Such competing constraints on protein function render brute force screens Ð used extensively by contemporary protein engineers Ð inefficient; therefore, new insights into navigating sequence space efficiently are always of interest. This past decade has seen the cost of reading and writing DNA drop precipitously 1. These advances have led to completely new ways to interrogate biology, including construction of thousands of synthetic genes 2, evaluation of functional effect o f tens of thousands of mutations in a protein in massively parallel experiments 3, sequencing of thousands of homologues across the tree of life for any given gene, and even ways to sequence human antibody repertoires 4. Such data -rich reservoirs are beginni ng to be tapped by protein engineers. ! 21 Here, we review recent progress in the use of data -driven protein engineering of potential therapeutics in order to satisfy multiple competing constraints. We restrict this review to protein engineering by amino acid substitutions. There are topical reviews concerning the de -immunization of protein therapeutics 5, engineering competing trade -offs in antibody function 6, and a comprehensive overview of engineered therapeutic enzymes 7. Thus, we will highlight representativ e and instructive examples utilizing de novo gene synthesis, deep mutational scanning, phylogenetic analysis, or anti body repertoire sequencing ( Figure 2.1 ); we also offer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igure 2.1: High throughput techniques used to overcome therapeutic protein engineering bottlenecks. A. Different bottlenecks encountered in developing therapeutics. B ÐC. Different high throughput techniques used fo r protein engineering. ! 22 perspective on future ways to harness the extraordinary torrents of data that we ant icipate in the coming decade. Large -scale mutational analysis Integrating deep sequencing with user -defined saturation mutagenesis 8 and screens such as yeast surface display sorting enables the determination of the functional effect of tens of thousands of mutations on a given protein sequence in a massively parallel fashion; this technique is known as deep mutational scanning 3. The mutational effect on each functional property of the protein can be assessed separately (if a screen exists), and mutations can be incorporated only if all screens give a positive result. Here, we give the latest examples of leveraging such datasets to engineer proteins while considering multiple constraints. Antibody deep mutational scanning Antibodies often bind two or more related proteins, and frequently the protein engineer is tasked with engineering specificity for one protein over the other. A recent notorious example involved the use of an antibody to monitor age -dependent levels of GDF11 in mice 9. However, this antibo dy also bound the closely related homolog GDF8 (myostatin), which resulted in erroneous conclusions 10 in the original paper. This multi -specificity problem is general, as many potential targets have very similar homologs in humans with some differing by on ly 10% or so in pairwise sequence identity 11-14; antibodies, therefore, must have exquisite specificity for therapeutic and diagnostic applications. Deep sequencing combined with a suitable screen can be used to identify specificity -modulating mutations i n protein binders 15. A recent excellent example comes from Koenig et al. ! 23 from Genentech 16, who sought to prevent binding to angiopoietin -1 (Ang -1) for a candidate antigen binding fragment (Fab) with desired binding to Ang -2 and vascular endothelial growth factor (VEGF). Using phage display, the authors determined the relative binding profile of many possible single point mutants in the six complementarity determining regions (CDR)s (483 light chain, 609 heavy chain) for each protein in parallel. From these 1092 mutations, 25 (2.3%) were shown in vitro to result in no or severely reduced binding to Ang -1. We note that all of these mutations slightly decreased binding affinities for Ang -2 and/or VEGF, illustrating the general difficulty Ð and in some cases, im possibility Ð of finding specificity -modulating mutations without a functional trade -off even when the local mutational space is comprehensively sampled. Thermal stability is another parameter that is often negatively correlated with binding affinity 17. Reduced Fab thermal stability results in lower expression titers 18. The necessity of engineering specificity -affinity while maintaining stability is critical for therapeutic application of binding proteins 19, and state of the art methods involve using co -screening for stability and affinity simultaneously 20. Alternatively, one can screen for stability and affinity in parallel to uncover rare, globally optimal mutations. Recently, the same Genentech team used this approach to scan all possible Fab frame work a nd CDR single point mutants for affinity and stable expression in a phage display context 21. Affinity -enhancing mutations were identified using selections against VEGF, while stabilizing mutations were identified with selections against protein A or protei n L. There were a handful of mutations improving both stability and affinity. In particular, mutation from Phe to Ala at a single framework mutation 25 † from the binding site, light chain residue 83 (LC -F83A), was found to strongly improve thermostability and affinity. The authors attributed this improvement to alteration of the interface between the variable and constant regions on the light chain, which was confirmed by hydrogen Ðdeuterium ! 24 exchange mass spectrometry. Intriguingly, LC -83 is one of the 5 Ð10% highest somatically mutated LC positions as determined by deep sequencing of over a thousand human lymphoid tissues. This suggests that LC -83 mutations can generally improve thermostability in Fabs, which the authors confirmed by incorporating LC -F83A in several unrelated mAbs. This is a great example of integrating naturally sourced deep sequencing data on a single target to obtain general insights into the functional tradeoffs of mutations in a broader set of antibodies. Enzyme deep mutational scanning Enzymes are applied in the treatment of diverse disorders such as cancer therapeutics to deplete essential amino acids or metabolic precursors needed by the cancer cells 22,23 , or as replacement therapies to return specific metabolites to healthier levels in a patient 24. Engineering non-immunogenic, serum -stable, and active enzymes can be challenging. For example, human kynurenine -degrading enzyme has low serum stability, and engineering has proven difficult because mutations that increase catalytic effici ency have decreased serum stability (J. Blazeck, personal communication). Understanding the trade -offs between stability and catalytic efficiency is critical for simplifying enzyme engineering. In contrast with protein binders such as antibodies, generali zable high -throughput screens for directly testing enzyme function do not exist. Thus, deep mutational scanning pipelines are not available for all enzyme classes. A creative solution for forward engineering stability and activity was presented by Klesmith et al., who presented the comparative analysis of deep mutational scanning datasets for solubility/stability and activity for two different model enzymes: levoglucosan kinase, and TEM -1 beta -lactamase25. From these datasets, Klesmith et al. were able to e xtract features common to mutations that improve stability while not hampering ! 25 catalytic activity. These mutations were sampled in the evolutionary history of the enzyme, were at least 15 angstroms from the active site, were in positions of the tertiary pr otein sequence without a large number of other residues in close contact, and were not mutations to or from proline. Combining these filtering metrics yields a greater than 90% probability of choosing a stabilizing, catalytically neutral mutation in any gi ven enzyme. This method has been developed into a Rosetta -based script and applied to increase the thermal stability and in vivo expression yield of a Type III polyketide synthase (E. Wrenbeck and T. Whitehead, unpublished results). Writing libraries of s ynthetic genes Compared with more limited datasets of proteins with 1 or 2 amino acid changes described above, datasets with wider tranches of sequence space may be more useful for identifying general engineering rules. For example, several enzyme discove ry efforts evaluate candidates by synthesizing hundreds of genes encoding enzymes spread throughout the protein superfamily 26,27 . Over the past five years the cost of writing DNA for kb -size genes has plateaued at about $0.10 per base pair 28, which means t hat libraries of several hundred genes can be written and tested on a medium -sized labÕs budget. A stunning example of the use of large protein datasets for obtaining engineering insights was recently described by Adimab scientists 18. Monoclonal antibodie s represent the largest class of engineered therapeutic proteins by revenue, with $98 billion in worldwide sales in 2017 29. Even with dozens of FDA -approved antibodies, some still fail in late -stage clinical trials for reasons unrelated to their binding af finity to their respective target. Understanding how a primary sequence determines manufacturability could help guide antibody -drug development akin to the Lipinski Ôrule of fiveÕ edict for small molecule drugs 30. To that end, Adimab scientists produced ! 26 a set of nearly all mAbs commercialized or in advanced stage clinical trials described in the patent literature through mid -2009 (137 in total) 18. They then assayed this set for a dozen biophysical properties including aggregation propensity, non -specific bi nding, and melting temperature. The first major insight was that Ð contrary to expectations Ð many of these antibodies had unfavorable scores in one or more measured biophysical property. The second major insight was the significantly better biophysical pr operties of the approved subset of mAbs compared to those in Phase 2 clinical trials, suggesting potential difficulties in translating some of these Phase 2 mAbs. Although this mAb dataset was only published in 2017, researchers are already mining it for predicting the sequence determinants of poor biophysical properties. For example, the Tessier group has used the Adimab dataset to find that positive CDR net charge correlates with mAb self-association using simple sequence -based scoring methods 31. This sa me group, following work on individual antibodies 32, has shown that reducing CDR net charge also decreases antibody nonspecificity (P. Tessier, personal communication). Going from hundreds to tens of thousands of proteins Ð with greater underlying predict ive power Ð requires a continued decrease in the cost of writing DNA. To that end, recent advances in DNA synthesis using microarray -derived oligo pools 1 have been utilized to synthesize thousands of designed small proteins 2,33 . Producing longer genes of 0 .8-2 kb in length from such oligo pools is difficult. The best approaches have a success rate of ~2% 34, and increasing the fidelity rate is essential for these emerging techniques to reach wider application. ! 27 Nature -sourced data Protein design by phyloge ny The cumulative sequencing data of thousands of protein homologs across the tree of life, for nearly all known therapeutically relevant proteins, are a rich resource for the protein engineer. It has been known for decades that, given any position in a p rotein family, the consensus residue is likely to be stabilizing 35. This insight enables the engineering of stable enzymes by simultaneously incorporating mutations at consensus positions in a protein sequence. An interesting variation on this idea comes f rom Nguyen et al., who used consensus sequence information to refine the specificity of an L -asparaginase from Erwinia chrysanthemi (ErA) 36. The therapeutic effect of wild type ErA is diminished by its dual activity as an L -glutaminase. While asparagine de pletion starves cancer cells, glutamine depletion is associated with many negative side effects 37. To reduce the L -glutaminase activity of ErA, conserved stretches of residues were identified within the active site. Nonconserved adjacent residues were targ eted for saturation mutagenesis with the assumption that such mutations could modulate specificity without disrupting desired catalytic function. This strategy resulted in a multi -point mutant with conserved L -asparaginase activity and a $25 -fold reduction of L -glutaminase activity. In vivo experiments using the engineered ErA have determined it is an effective therapy for both T and B-cell acute lymphoblastic leukemia without the side effects of glutamine depletion 38. Consensus mutations informed by phylo geny are increasingly incorporated into structure -based computational design algorithms to engineer stabilized proteins 39. To cite as the best example, Goldenzweig et al. developed a Rosetta -based method to stabilize human enzymes 40. Evolutionary conservat ion data were converted to a position specific scoring matrix, and only mutations above a certain conservation threshold were considered. Mutations passing additional ! 28 structure -based filters were combined into new designs. They used this approach to increa se the expression yield and stability of a human acetylcholinesterase variant, which can be used for organophosphate detoxification following nerve agent exposure. Use of this method led to an acetylcholinesterase variant with 51 mutations, $2000 -fold incr eased bacterial expression compared to the original enzyme, and with nearly the same specific activity. The consensus mutation approach has reached its logical end -state with the use of ancestral sequence reconstruction to improve protein function 41-43. Resurrecting ancient proteins often leads to enzymes with incredible stabilities 44. Zakas et al. used this general understanding to resurrect several ancestral Factor VIII proteins 45. By doing so, they engineered a therapeutically effective ancestral Factor VIII variant with higher stability than extant forms. Immunogenicity is always a major concern for candidate therapeutic proteins. For example, a highly engineered Factor VII variant failed late stage clinical trials because of immunogenicity 46. Interest ingly, the reconstructed ancestral Factor VIII variants share 95% sequence identity with human Factor VIII, but have reduced cross -reactivity with known anti -human Factor VIII antibodies 45. This reduced cross -reactivity was verified in in vitro analyses. I t will be interesting to see if these in vitro experiments translate to lower immunogenicity in animal models, and whether ancestral proteins have lower immunogenicity than extant proteins in general. Engineering from human antibody repertoires We are in an exciting age of molecular serology, moving on from bulk measurements of humoral responses towards descriptions of full sequences and functions of individual antibodies in human repertoires 47-49. These repertoire sets are a critical resource for underst anding affinity ! 29 maturation, CDR development, and pairing between heavy and light variable chains (V HÐVL). While there has not been a great deal of engineering work in the open literature exploiting these repertoire datasets, we expect that to change in the next few years. The most recent use of information from these repertoires was described from Adler et al. 50, who showed that incorporating native V HÐVL pairing improved the quality and quantity of candidate anti -interleukin 21 receptor antibodies isolated from a yeast display screen. Anticipated use of these datasets include designing more efficient antibody libraries by incorporating information from CDR lengths and net charges, and position specific substitution patterns. To facilitate this goal, Sheng e t al. constructed gene -specific substitution patterns for 69 common V genes 51, while Kovaltsuk et al. have collected nearly all repertoi re datasets into a single data -base called the Observed Antibody Space 52 (antibodymap.org). Perspective The ability to write a nd read DNA at scale has trans formed protein engineering into a big data Figure 2.2: *Big data yields insights for efficient engineering of protein therapeutics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field. Here, we have discussed a diversity of data -rich methods for improving protei n therapeutics, with each of these methods providing unique engineering insights ( Figure 2.2 ). In the next few years, we anticipate further ways to leverage and combine data from multiple sources to improve protein engineering. One of the key emerging idea s is to enhance protein design by incorporating constraints from the evolutionary history of the protein. These constraints are mostly identification of conservation at a single position, but for human antibodies with millions of sequences we imagine more sophisticated algorithms that take into account correlation between residues within or across CDRs, bulk biophysical properties like CDR net charge, and sequence properties as a function of CDR loop length. We also anticipate a marriage of data analysis fr om a post -hoc view by combining nature -sourced data of existing protein sequences with the forward -evolutionary view of deep mutational scanning to observe the range of possible mutations for a given candidate therapeutic. This integration will allow a com prehensive look at Ôwhat isÕ with Ôwhat ought to beÕ, enabling the protein engineer to design functional proteins on demand. Acknowledgements This work was supported by the National Science Foundation [Career Award #1254238 CBET to T.A.W] and Michigan St ate University [Johansen Crosby endowed chair to T.A.W.]. ! 31 REFERENCES ! 32 REFERENCES 1. Hughes RA, Ellington AD: Synthetic DNA synthesis and assembly: putting the synthetic in synthetic biology . Cold Spring Harb Perspect Biol 2017, 9:a023812. 2. 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J Immunol 2018, 201:2502 -2509. !!!!!!!!!!!!!!!!!!! 37 CHAPTER 3 Impact of in vivo protein folding probability on local fitness landscapes This chapter is adapted with permission from the publication ÒImpact of in vivo protein folding probability on local fitness landscapes Ó in Molecular Biology and Evolution by Matthew S. Faber, Emily E. Wrenbeck, Laura R. Azouz, Paul J. Steiner, and Timothy A . Whitehead. Copyright 2019 Oxford University Press . ! 38 Abstract It is incompletely understood how biophysical properties like protein stability impact molecular evolution and epistasis. Epistasis is defined as specific when a mutation exclusively influences the phenotypic effect of another mutation, often at physically interacting residues. In contrast, nonspecific epistasis results when a mutation is influenced by a large number of non -local mutations. As most mutations are pleiotropic, the in vivo folding probability - governed by basal protein stability - is thought to determine activity -enhancing mutational tolerance, implying that nonspecific epistasis is dominant. However, evidence exists for both specific and nonspecific epistasis as the prevalent factor, with limited comprehensive datasets to support either claim . Here we use deep mutational scanning to probe how in vivo enzyme folding probability impacts local fitness landscapes. We computationally designed two different variants of the amidase AmiE with statistically indistinguishable catalytic efficiencies but lower probabilities of folding in vivo compared to wild -type. Local fitness landscapes show slight alterations among variants, with essentially the same global distribution of fitness effects. However, specific epistasis was predominant for the subset of m utations exhibiting positive sign epistasis. These mutations mapped to spatially distinct locations on AmiE near the initial mutation or proximal to the active site. Intriguingly, the majority of specific epistatic mutations were codon -dependent, with diff erent synonymous codons resulting in fitness sign reversals. Together, these results offer a nuanced view of how protein folding probability impacts local fitness landscapes, and suggest that transcriptional -translational effects are as important as stabil ity in determining evolutionary outcomes. ! 39 Introduction Understanding the mechanisms of molecular evolution is important to molecular biology, virology, evolutionary biology, and protein engineering. Researchers interested in evolving natural proteins, designing proteins de novo , or understanding the extent of contingency on extant proteins must contend with the implicit evolutionary limitations set forth by nature. The challenge, then, is to understand what constrains protein evolution and by what mech anisms. How do these factors interact with one another to alter the frequency of mutations with increased fitness in a given environment, and how do they govern evolvability for new functions? A particularly important component of evolution is epistasis, or the non -additive combination of mutations 1. Epistasis impacts the rate of evolution and the spectrum of possible evolutionary pathways available to a protein 2. Epistasis is said to be specific when a mutation exclusively influences the phenotypic effec t of only a few select mutations, usually at physically interacting residues 3. In contrast, epistasis is said to be nonspecific when a mutation impacts a global property like stability that can be rescued by large numbers of non -local mutations. Of the two classes, specific epistatic effects exert the greatest influence on the possible evolutionary outcomes 3. This is the result of the precise and long -lasting amino acid constraints imposed by specific epistatic mutations, which decrease the evolutionary rev ersibility of a protein sequence in a given evolutionary trajectory. In turn, the delocalized and unconstrained effects resulting from non -specific epistasis often breakdown over evolutionary time, largely restricting its influence to short -term evolution. Non -specific epistasis can temporarily alter the mutational robustness of a protein by changing the permissivity to additional mutations at many more positions than does specific epistasis. By modulating the evolutionary trajectories available, epistatic phenomena exert immense influence on the short and long -term evolution of proteins 4. ! 40 What remains incompletely understood is how biophysical parameters like protein stability constrain epistasis . Protein stability as defined here is the cumulative balan ce of the thermodynamic stability, the folding rate, and the fidelity of the association process for oligomeric proteins; these parameters combine to determine the likelihood that an enzyme will assume its native state when expressed in vivo : the in vivo folding probability of a given protein 5,6 . For enzymes, fitness is often a function of flux through a pathway, which is a product of steady state enzyme concentration and specific velocity. The steady state enzyme concentration, in turn, is a function of the translation rate, the probability of the nascent peptide folding into the native state, and the protein degradation rate. Typical evolutionary models control for translation rate and protein degradation rate and then assume that (i.) thermodynamics of protein folding can be described by a 2 -state model; and (ii.) this single Gibbs free energy term can account for the probability of folding. However, these assumptions fail for much of a typical proteome. Many proteins are oligomeric, multi -domain protein s have more complicated folding trajectories, and there is increasing evidence that formation of secondary structure and partial hydrophobic collapse before ribosomal release is important for on -target folding 7. The in vivo folding probability encompasses all of these biophysical terms into the probability of reaching the folding state. Analyses of the impacts of stability in evolution at the genomic 8, protein 9-11, and organismal 12 levels have uncovered a complex and dynamic equilibrium between stabilizing and destabilizing mutations. For enzymes in particular, previous studies have shown that missense mutations often act pleiotropically where catalytically enhancing mutations are, on average, moderately destabilizing 9,13,14 . Consequently, high basal stabil ity can buffer catalytically beneficial but destabilizing mutations 15,16 , allowing fixation. Deleterious destabilizing mutations ! 41 can be repaired by reversion mutations 17, or by specific and non -specific epistatic mutations that rescue stability 8,18 . These epistatic mutations are a central phenomenon in the stabilizing -destabilizing equilibrium, with significant consequences in long -term evolution 19,20 . It is uncertain whether specific or non -specific epistatic mutations are more likely to rescue a destabili zed protein, with evidence existing for both arguments 17-21. Deep mutational scanning experiments provide a wealth of mutational data that can be used to address questions in molecular evolution 22. This technology comprises the use of large mutational li braries with selections coupled to deep sequencing to evaluate relative fitness of thousands of variants in a massively parallel fashion 14,23 -25. We previously used deep mutational scanning on the homohexameric aliphatic amidase AmiE from Pseudomonas aerug inosa to understand how local fitness landscapes, defined here as the set of all possible single -point amino acid substitutions from wild -type (WT), change with different substrates 25. In this original study, AmiE was chosen as a model as it is stable in i ts genetic background and has a high probability of folding upon translation. To comprehensively assess how the initial probability of folding in vivo constrains mutational outcomes, we designed two variants of AmiE in which catalytic activity is unperturb ed but the proteins have different in vivo folding probabilities. We then used deep mutational scanning to probe the local fitness landscapes of these variants. While we found moderate epistasis, local fitness landscapes are largely insensitive to the init ial in vivo folding probability of the enzyme variant. In particular, the great majority of beneficial mutations were shared between all three starting points: WT AmiE, and the two disrupted single point -mutant enzymes. However, positive sign epistasis was present and was dominated by specific epistasis. Remarkably, we found that the sign of the fitness metric for many mutations depends on the codon used to encode the mutation, suggesting more complicated fitness landscapes than ! 42 predicted from intrinsic pro tein biophysics. Together, these results provide a nuanced view of how local fitness landscapes are perturbed under slightly different initial in vivo folding probabilities. Results The experimental pipeline used in this study is shown in Figure 3.1A . First, we designed variants of AmiE that possess wild -type catalytic activity but with a reduced probability of folding in vivo. Second, we developed selection conditions for the variants under which cell growth is proportional to enzyme activity using a growth selection with acetamide as the sole nitrogen source. Third, near -comprehensive single -site saturation mutant libraries for our variants were prepared 25 and growth selections performed. Fourth, pre - and post -selection populations were deep sequenc ed to extract mutant frequencies in the selected and reference populations. These frequencies were converted into a relative fitness metric ( !i) for each mutant i defined as !!!!"#!!!!!!"#! (1) where !i and !REF represent the specific gro wth rates in selection media for the mutant (µ i) and unmutated AmiE variant (µ REF ), respectively. A relative fitness score above zero means that a strain harboring a given mutant has higher fitness than those carrying the unmutated variant. It is important to understand the limitations of deep mutational scanning experiments. While these experiments provide quantitative measurements of fitness relative to the respective backgrounds, deep mutational scanning cannot provide information on why a given mutation is beneficial or deleterious. Thus, specifying whether a mutation impacts stability, catalytic efficiency, on -path folding rate, etc. is left to reasoned speculation or further biophysical analysis. ! 43 AmiE variants with lower in vivo folding probabilities a nd wild -type catalytic efficiencies engineered We first sought to identify mutations to AmiE that, under the selection conditions, would decrease the in vivo folding probability of the protein while maintaining wild -type catalytic efficiency. To identify such mutants, we chose to use a computational approach by modifying PROSS 26. Briefly, PROSS designs a protein sequence that will have an improved probability of reaching the folded state in vivo relative to its input. This improved folding probability corr elates with biophysical properties like improved protein stability, faster on -target folding rate, or reduced aggregation propensity. As our experimental objective is essentially the inverse problem, we modified the Rosetta FilterScan protocol undergirding PROSS and then selected point -mutations with higher energy scores relative to AmiE wild -type (WT) ( Figure 3.1B, Table A 1). For each mutant these scores were then cross -referenced with experimental relative fitness scores previously determined for AmiE 25 to ensure that their relative fitness was below zero (Table A 1). Of thirteen variants with 1 -3 mutations from WT selected for experimental characterization, nine expressed as soluble proteins in E. coli BL21* (DE3). We purified a subset of these nine v ariants and assessed their catalytic efficiency with the substrate acetamide. While most mutants showed reduced enzymatic activity, both AmiE I38V and AmiE I122L showed statistically indistinguishable maximum turnover rates (kcat) and Michaelis constants ( KM) compared with WT ( Figure 3.1C, Table A 2). Furthermore, size exclusion chromatography showed no oligomeric differences between AmiE WT and AmiE I38V or AmiE I122L ( Figure ! 44 !"#$% !"#$%&'(&)*+"' ,&-&./0'+1/234' )&.&53*/"'5/"(*3*/")' É!!!&!'('!&(& É%É!(' !!!('!&(& É%É!('&!' !!!&(&É%É!('&!'('! !!!É%6*7181$'+&"&183*/"' 8"('+1/234')&.&53*/"' 95&38%*(&':1/234' ;&.&53*/")' ,&&0';&<=&"5*"+'8"('8"8.$)*)' >=383*/")'3483' *%08*1'?/.(*"+'8"(' (/'"/3'*%0853' 5838.$3*5'853*-*3$' @"51&8)*"+'?/.(*"+' 01/787*.*3$' !"#$!$%# ')(%!"#$% A*3"&))'.8"()580&)' A*3"&))'!??&53' *%+%,%-.//0% -123% B&..' +1/234' ;*"+.&')*3&')83=183*/"' %=38"3'.*7181*&)' ;387.&' @%08*1&(' 95&38%*(&' C8))*-&' (*??=)*/"' !"#$% DEF'G'&'#(%)!# B$3/)/. #0.000.050.100.150.200.25wt_refolded wt_unfolded i122l_refolded i122l_unfolded i38v_refolded i38v_unfolded Sampleactivity11121314151617wt_refolded wt_unfolded i122l_refolded i122l_unfolded i38v_refolded i38v_unfolded Sampleactivity!"!#$" !#%" !#&" !#'" ("(#$" )*+,-,./"0,.1/" 23+451,6./"73"89" !#!" !#$" !#%" !#&" !#'" (#!" (#$" 89" :;'<" :($$=" >"?";" @"A"!"#"$%&'()*"#"+,+++-" ."#"$%&'()*"#"+,++/" Variant !"#$%&'"() *$% ()*$% (+,-$#."( !"#$%&'"(/ 0(/0(+,-$#."( AmiE I122L 1.08 ± 0.06 0.57 0.89 ± 0.15 0.64 AmiE I38V 1.02 ± 0.08 0.79 0.82 ±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urface occluded enzyme core 90¡ 90¡ Surface occluded enzyme core !"!#$" %"%#$" &"&#$" !"$"%!"%$"&!"'(" )*+," !"!#$" %"%#$" &"&#$" !"$"%!"%$"&!"'(" )%&&-" !"#$%&'()*+,-./"%0) !"#$%&'()*+,-./"%0) ./0123450"6378" ./0123450"6378" !"# $%&&'#$()*#.6789:;<#)8=>(7:?6*#;8# @87*6*#6)?<>6#ABC# D"D# D"DE# D"/# D"/E# /0#/,#/F#/E#/G#/H#Figure 3.1: Design of deep mutational scanning experiment A. A graphical overview of this study. Two AmiE enzyme variants with single point mutants (I38V and I122L) with WT catalytic function and lower probabilities of folding in vivo were computationally designed and validated experimentally. Constitutive expressio n of each enzyme from a plasmid was tuned such that the growth rate of our bacterial growth selection strain in selection media was dependent on the expression of functional AmiE. Deep mutational scanning was performed on these variants and compared with W T AmiE. B-G. Design and validation of AmiE variants. B. A graphical representation of the computational enzyme design. C. Enzyme velocity as a function of acetamide and Michaelis -Menten parameters determined relative to WT AmiE. Error bars = 1 s.d., n = 2, p-values obtained using StudentÕs t -test. D. Structural modeling of the cavities introduced into AmiE by designed mutations. E. Enzyme yield following E. coli auto -induction expression. Error bars = 1 s.d., n = 3, * = p -value = 0.0003, # = p -value = 0.00 2. F. Specific growth rates of strains in M9 (unselective) and in M9 with 10 mM acetamide as sole nitrogen source (selective) (pEDA2 - low expression, pAG - high expression). Error bars = 1 s.d., n " 3. G. Comparison of enzyme reaction velocities at substr ate saturation relative to a folded control. Grey dots represent biological replicates, Error bars = 1 s.d., n = 2. ! 45 A 1) in PBS at 30 µM, suggesting that the variants maintain the expected homohexameric quaternary structure. Finally, the secondary structu re of the three AmiE proteins was analyzed using far -UV circular dichroism spectroscopy ( Figure A 2), revealing indistinguishable spectra in the folded and unfolded states. AmiE I38V removes a methyl group to open a small cavity in the core, while I122L modulates hydrophobic core packing in the monomer subunit ( Figure 3.1D, Figure A 3). Both mutations are located in the hydrophobic core distal from the dimeric and homohexameric contacts necessary for quaternary assembly ( Figure A 3). Based on Rosetta analysis, we predict that these mutations disrupt the core of AmiE resulting in thermodynamic destabilization of the native monomer. However, mutations in the stability cores of proteins can disrupt the hierarchy of folding 27 through the destabilization of folding intermediates 28, by limiting the intermediate states accessible during folding 29, and by decreasing the thermodynamic stability 30 of the monomeric subunits outside of the quaternary structure 31. To distinguish among these possibilities, we attemp ted tryptophan fluorescence unfolding measurements using guanidinium -HCl (Gdn -HCl) as a denaturant to determine the effective thermodynamic stability. However, AmiE WT aggregated in moderate Gdn -HCl concentrations under most conditions (data not shown), an d under conditions of no aggregation and complete unfolding no isosbestic point was recovered ( Figure A 4). This lack of an isosbestic point indicates more complicated reversible folding at 4¡C than simple 2 -state models. We also performed thermal shift as says with the purified homohexameric enzymes in a series of dilutions (10, 5, 1, 0.5, 0.25 µM) ( Figure A 5) to measure thermal stabilities. Two -state irreversible unfolding curves were obtained at 10 and 5 µM. Analysis of the melting curves reveals statistically indistinguishable melting temperatures between WT and variants ( Table A 3). This ! 46 data suggests that all have a single transition from homohexamer into unfolded monomers. It is well established that oligomeric proteins are often more stable than in t heir natively folded monomeric or dimeric forms 31. Thus it is likely that the thermal melt is measuring the stability of homohexameric assembly, which would be expected to be identical between WT and variants as neither mutation resides at an oligomeric in terface. To identify AmiE concentrations for which the monomeric form is favored, we reasoned that hexameric dissociation would result in inactive enzyme 32, which could be measured colorimetrically using our established activity assay. Indeed, activity ana lysis of dilute solutions of AmiE WT at 500 nM showed larger decreases in activity than 900 nM over moderate incubation periods ( Figure A 6). Unfortunately, usable signal was not detected at protein concentrations of less than 5 µM in the thermal shift ass ays ( Figure A 5). To assess thermal denaturation at lower enzyme concentrations, we performed circular dichroism thermal melts using a protein concentration of 1 µM ( Figure A 7). Under these conditions both AmiE I38V and AmiE I122L have modest but signific antly lower melting temperatures than AmiE WT (p -value 0.01 for I38V and 0.03 for I122L; Table A 4). While we were only able to establish moderate decreases in the thermal stabilities of the AmiE variants, complementary in vitro and in vivo experiments st rongly support that both I122L and I38V variants have lower in vivo folding probabilities than WT in the general order: I38V6x10 6 cells for approximately 8 generations at 37¡C. A biological replicate for AmiE WT covering residues 171-255 was also performed to compare with previous published results 25. The pre - and post -selection populations were barcoded and deep sequenced. The resulting data was processed using PACT 35 to obtain the relevant fitness metrics for each mutant in the library. The depth of sequencing ranged from 155 to 300 -fold coverage for the libraries ( Figure A 8). In total, we rec overed the relative fitness metrics for 93.7% and 91.8% of all possible non -synonymous mutants for AmiE I122L and AmiE I38V, respectively ( Table A 7). To estimate reproducibility, we compared the AmiE WT replicate selections performed here with data from an identical selection experiment performed in Wrenbeck et al. 25. Correlation coefficients between mutation -specific fitness values in selections are "0.90 (Figure A 9), which is comparable to correlation between replicates performed for this work (AmiE I122L - 0.921; AmiE I38V - 0.952) (Figure 3.2A ). Additionally, there was essentially no correlation be tween relative fitness and pre -selection frequency of a given mutant in the library, (WT AmiE Ð R = 0.011, AmiE I122L Ð R = 0.026, AmiE I38V Ð R = -0.0376) (Figure A 10) indicating that pre -selection read counts do not bias the fitness metrics obtained. ! 49 Distribution of beneficial fitness effects are largely insensitive to initial in vivo protein folding probability The shape of the distribution of fitness effects (DFE) governs the local protein fitness landscape. Realizing that beneficial mutations are rare, the likelihood of finding beneficial mutations was predicted by Orr 36 to follow the Pareto family of distributions. Using the set of beneficial mutatio ns Ð variants with relative fitness above wild -type under selective media Ð we were previously able to describe the shape of the DFE for beneficial mutations as exponential with high statistical power 25. The new datasets allow us to ask directly whether th e shape of DFE changes with respect to enzyme in vivo folding probability. Consistent with expectations, all variants have very similar distributions of fitness effects ( Figure 3.2B and Figure A 11) with a tight range of total possible mutations that are b eneficial. For all variants the Pareto family of functions also describes their distributions of beneficial fitness effects ( Table A 8). Thus, given approximately the same relative fitness, the probability of finding rare beneficial mutations is independent of initial likelihood of native folding. ! 50 !"#$"%"#&!"#'"()"*"+,"-./0" -./0"1(*2" -./0"11$$3" 0 102030405060!0.0250.0250.0750.1250.1750.2250.2750.3250.3750.4250.4750.5250.5750.6250.6750.7250.7750.8250.8750.9250.975BinI38V_bene_total0 102030405060!0.0250.0250.0750.1250.1750.2250.2750.3250.3750.4250.4750.5250.5750.6250.6750.7250.7750.8250.8750.9250.975xy!"#$"#%"#&"#'"#("#"#"#")'# !#0 102030405060708090!0.0250.0250.0750.1250.1750.2250.2750.3250.3750.4250.475BinI122L_bene_total0 102030405060708090!0.0250.0250.0750.1250.1750.2250.2750.3250.3750.4250.475xy!"#$"#%"#&"#'"#("#)"#*"#+"#"#"#",'# ",$'# 0 10 20 30 40 50 60 70 80 90 100110!0.0250.0250.0750.1250.1750.2250.2750.3250.3750.4250.4750.525BinWT_bene_total0 10 20 30 40 50 60 70 80 90 100110!0.0250.0250.0750.1250.1750.2250.2750.3250.3750.4250.4750.525xy!"#$"#%"#&"#'"#("#)"#*"#+"#!""#!!"#"#"#",'# ",$'# 0 100 200 300 400 500 30003100320033003400!0.95!0.85!0.75!0.65!0.55!0.45!0.35!0.25!0.15!0.050.050.150.250.350.450.550.650.750.850.95BinI38V_nonsense0 100 200 300 400 500 30003100320033003400!0.95!0.85!0.75!0.65!0.55!0.45!0.35!0.25!0.15!0.050.050.150.250.350.450.550.650.750.850.95BinI38V_nonsense!"#!!"$!!"%!!"&!!"'!!"%%!!"%$!!"%#!!"%!!!"!"!('" #")!('"0 100 200 300 400 500 28002900300031003200!1.05!0.95!0.85!0.75!0.65!0.55!0.45!0.35!0.25!0.15!0.050.050.150.250.350.45BinI122L_nonsense0 100 200 300 400 500 28002900300031003200!1.05!0.95!0.85!0.75!0.65!0.55!0.45!0.35!0.25!0.15!0.050.050.150.250.350.45BinI122L_nonsense!"#!!"$!!"%!!"&!!"'!!"%#!!"%!!!"$(!!"$)!!"!"!*'" +!*'"0 100 200 300 400 500 22002300240025002600!1.45!1.35!1.25!1.15!1.05!0.95!0.85!0.75!0.65!0.55!0.45!0.35!0.25!0.15!0.050.050.150.250.350.450.55BinWT_nonsense0 100 200 300 400 500 22002300240025002600!1.45!1.35!1.25!1.15!1.05!0.95!0.85!0.75!0.65!0.55!0.45!0.35!0.25!0.15!0.050.050.150.250.350.450.55BinWT_nonsense!"#!!"$!!"%!!"&!!"$'!!"$&!!"$%!!"!"!('" )!('")#"! WT ! I122L ! I38V !"#$%&'()'*+&,+-./' Decay = -3.62 Decay = -9.32 Decay = -9.56 !(-/%-/%' 0,//%-/%' !(-/%-/%' 0,//%-/%' !(-/%-/%' 0,//%-/%' !0.50.00.51.0!1.0!0.50.00.5WT AmiEI38V AmiE!1.0!0.50.00.5!0.50.00.51.0I38V AmiEI122L AmiE-0.5!0.0!0.5!1.0!I38V Fitness Metrics!-1.0!-0.5!0.0!0.5!I122L Fitness Metrics!-1.0!-0.5!0.0!0.5!WT Fitness Metrics!-0.5!0.0!0.5!1.0!I38V Fitness Metrics!!1.0!0.50.00.5!1.0!0.50.00.5WT AmiEI122L AmiE-1.0!-0.5!0.0!0.5!WT Fitness Metrics!-1.0!-0.5!0.0!0.5!I122L Fitness Metrics!!0.8!0.40.00.4!0.8!0.40.00.4Fitness_wt_tr1Fitness_wt_tr2WT!-0.4!0!0.4!-0.8!-0.4!0!0.4!-0.8!Fitness Metric Replicate 1!Fitness Metric Replicate 2!!0.50.00.51.0!0.50.00.51.0Fitness_i38v_tr1Fitness_i38v_tr2I38V!1!0.5!0!0!0.5!1!Fitness Metric Replicate 2!Fitness Metric Replicate 1!-0.5!-0.5!!0.8!0.40.00.4!0.8!0.40.00.4Fitness_i122l_br2_tr2Fitness_i122l_br2_tr2I122L!-0.8!0.4!0!0!-0.4!-0.8!0.4!-0.4!Fitness Metric Replicate 2!Fitness Metric Replicate 1!!"#$%&%$ !"#$! '(')*"#$%&%$+ !%&'()(*+! '(,#!+%(')"'('! -)".(/()0! .122+3+$! .+#+2(4("/! 51)")(&#'! 67!! 87!! 97!!:7!!R=0.79 R=0.72 R=0.79 R=0.93 R=0.92 R=0.95 Figure 3.2: Local fitness landscapes are nearly insensitive to initial protein folding probability in vivo . A. Correlation between AmiE variant technical replicates. B. Distributions of fitness effe cts (DFE) of nonsense and missense mutations for the AmiE variants. Upper plots show full DFE, while lower plots include only beneficial mutants with best -fit exponential curve. C. Correlation of fitness between AmiE variants following the combination of replicate datasets and reprocessing using PACT. D. Venn diagram for all unique and shared beneficial mutations for the respective variants. ! 51 Moderate epistasis observed with decreasing enzyme in vivo folding probability How does the local fitness landscape change in response to a single deleterious point -mutation that alters only the in vivo folding probability of an enzyme ? If mutations were completely additive with the disrupting mutations, we would expect the comparison of the local fitness landscapes for the enzymes to have 1:1 correlations and approach the correlation coefficients found between replicates (R ~0.92). On the other hand, complete non -additivity of mutations would lead to minimal correlation. We were able to compare 2,813 mutations above the lower bound of relative fitness (45.4% of possible mutations) shared between the three datasets. PearsonÕs correlation analysis of the DFE finds that the WT local fitness landscape is reasonably correlated with that of the variants (WT vs. I38V R= 0.72 , WT vs. I122L R = 0.79), and this correlation is similar to that between I122L vs. I38V (R= 0.79) ( Figure 3.2C ). Notably, these correlation coefficients are lower than for replicates. Furthermore, linear regression best fits show lower slopes between vari ants than within replicates ( Figure 3.2A and 3.2C). Next, we tested for increased negative epistasis in our distribution of fitness effects. We predicted that the greater in vivo folding probability of AmiE WT provides a buffering effect, which could tem per many deleterious mutations. To test this hypothesis, we generated empirical cumulative distribution functions (ECDF) for the deleterious mutations for each of the three enzymes ( Figure A 12 and Table A 9). This analysis is limited to the range of delet erious mutations quantitatively captured in our experimental system 23. Within this range, the application of the Kolmogorov -Smirnoff test failed to reject the null hypothesis that the ECDF of AmiE WT is below that of AmiE I38V ( Table A 9). By the same test , we were unable to discriminate the ECDFs of AmiE WT and AmiE I122L ( Table A 9). Therefore, AmiE I38V has a greater number of more deleterious mutations than AmiE WT. This indicates that the lower in ! 52 vivo folding probability of the AmiE I38V background in creases the likelihood of negative epistatic effects when compared to the more resilient AmiE WT. Precise measurements of negative and positive epistasis are complicated by the relatively narrow range of fitness our experimental system captures. However, we can determine the sign of fitness in our datasets with high precision. To evaluate the relative prevalence of sign epistasis, we defined any mutant as beneficial if !i > 0 for both replicates and if !i > 0 within a 95% confidence interval (see Materia ls and Methods ). Conversely, we define a mutant as deleterious if !i < 0 for both replicates and if !i < 0 within a 95% confidence interval. We used these cutoffs to sort beneficial variants into the seven possible fitness bins ( Figure 3.2D ). Using a str icter requirement - that beneficial mutants are defined as those with a "10% increase in specific growth rate over the genetic background - leads to similar results ( Figure A 13). Most beneficial mutations in the WT background are shared Previous studies found that stable proteins can buffer destabilizing mutations that are otherwise beneficial 37,38 . We are able to assess the extent of this phenomenon in our datasets. We find that 122/141 (86.5%) of beneficial mutations in the WT background are also benef icial in the I38V and/or the I122L genetic background ( Figure 3.2D ). Of these, six globally beneficial mutations (S9A, A28R, R89E, I165C, V201M, A234M) have been previously characterized biophysically 25 and are known to improve specific amidase flux under the selection conditions of 10 mM acetamide at 37¡C. Conversely, only 19 of 141 beneficial mutations (13.5%) are specific in the WT background ( Figure 3.2D ). Therefore, beneficial mutations that are buffered in the stable background are present but in the minority. ! 53 The 19 WT -specific beneficial mutations map to two predominant locations: eleven (G291C, L297H, D311C, E320A, S325Y/T/C, 326Y/H, R336L, G341Y; Figure 3.3A ) are at the extreme C -terminus that creates extensive homodimer contacts, while four (M72T , E74G, A78H, E82I; Figure 3.3A ) are located on helix B and helix C distal to any oligomeric contacts in the homohexamer. The majority of the C -terminal mutations are adjacent to the homodimerization interface, which we speculate could lead to subtle struc tural rearrangements in the AmiE active site. Probable mechanisms behind the helix B/C mutations are more obscure as three of these mutations are at surface exposed positions over 10 † away from any active site residue. We note that active site -induced ef fects may still be quite strong even this far away 14,25,39 -41. Regardless of the exact mechanisms, these findings indicate localized regions where small -scale mutational perturbations lead to increased fitness in a more stable genetic background. To avoid information loss from the simple categorical separation of mutations into bins, normalized linear regression analysis was performed on correlation plots for the beneficial mutations shared in all backgrounds, in which the best -fit linear regression for th e respective correlation plots was determined and normalized such that Y = 1X + 0 (Figure A 14). In these correlation plots the mutational effects are highly disperse and widely deviate from the predicted fitness metric function for non -epistatic mutationa l combinations. The regression normalized AmiE I38V (Figure A 14A) and AmiE I122L ( Figure A 14B) datasets compared with WT both show decreased correlations (AmiE I38V PearsonÕs R = 0.62; AmiE I122L PearsonÕs R = 0.66) compared to within replicates (Pearson Õs R >0.92). These distributions indicate that both classes of epistatic effects are present and imposing large impacts on this population of beneficial mutations. ! 54 Figure 3.3: Positive sign epistatic mutations are spatially segregated and specific . A-C. Bar graphs of fitness metrics. Error bars represent 95% confidence intervals calculated from Poisson errors inherent in deep sequencing (Klesmith et al. 2015), while grey dots are fitness metric for each replicate. Horizontal dotted lines represent the cutoff value for mutations that increase the growth rate by "10%. Models show: trimer of dimers (wire + surface models), background residues for a respective enzyme (white mesh + sticks), the active site residues (magenta spheres), and where applicable the original mutation (green or orange mesh + sticks). In AmiE I122L and AmiE I38V the unique beneficial mutations tend to cluster around the disrupting mutations or near the active site. A. AmiE WT unique beneficial mutations are located at the C -terminal tail or in the B/C helices. B. Location of AmiE I122L unique beneficial mutations segregate to either positions adjacent to position 122 (T84I/V) or adjacent to the active site. C. Locations of a subset of AmiE I38V unique beneficial mutations. In B and C the blue transparent surface with sticks represents the residue in AmiE WT, and green or orange transparent surfaces with sticks represent respective unique beneficial mutations. !"#$%&'()% *+,-./0123% /45#314% &'()%3#5/167#2.% "17+7#02% !"#$%89% /45#314% &:;;<%%3#5/167#2.% "17+7#02% !"#$%&:;;<% *+,-./0123% /45#314% !"#$%89% /45#314% !"#$%89% *+,-./0123% /45#314% !"#$%89% *424=#,#+>% "17+7#02% !,7#?4%5#74% /45#314% !"#$%&:;;<% *424=#,#+>% "17+7#02% !,7#?4%5#74% /45#314% !"#$%&'()% *424=#,#+>% "17+7#02% !,7#?4%5#74% /45#314% !"#$%&'()*+,'-.)//+0)-#'1/+ 2345+!0.6!0.30.00.30.621P_i38v21P_wt21P_i122l25C_i38v25C_wt25C_i122l102I_i38v102I_wt102I_i122l251N_i38v251N_wt251N_i122l253Y_i38v253Y_wt253Y_i122l254T_i38v254T_wt254T_i122l65V_i38v65V_wt65V_i122l143T_i38v143T_wt143T_i122l190T_i38v190T_wt190T_i122l193P_i38v193P_wt193P_i122l230C_i38v230C_wt230C_i122lMutationfitness2346+3+346+ 345+ 789:+;'/#<=-'.>+$<-%-'".+%*?%1).-+ @1-'A)+/'-)+%*?%1).-+ I38V L102I Q254T I253Y M251N K21P L25C Q190T F230C M193P G143T I65V B8CD+ B938E+ 08C9!+ E8C6F+ G8CHI+E5CJ+ K9H6I+G9L3I+09L6:+ ,863D+ @1-'A)+/'-)+%*?%1).-+ !"#$% !&''(%)*%;'/#<=-'.>+$<-%-'".+%*?%1).-+ !1.0!0.9!0.8!0.7!0.6!0.584i_i122l84i_wt84i_i38v84v_i122l84v_wt84v_i38v58h_i122l58h_wt58h_i38v69q_i122l69q_wt69q_i38v136v_i122l136v_wt136v_i38vMutationfitness!0.45!0.25!0.050.1584i_i122l84i_wt84i_i38v84v_i122l84v_wt84v_i38v58h_i122l58h_wt58h_i38v69q_i122l69q_wt69q_i38v136v_i122l136v_wt136v_i38vMutationfitness!"#$%&'()*+,'-.)//+0)-#'1/+ 23435+23465+3475+ 2348+9:;<+ ='/#>?-'.@+$>-%-'".+%*A%1).-+ 9:;B+ C5:D+ CEFG+<7HEB+ I1-'J)+/'-)+%*A%1).-+ ='/#>?-'.@+$>-%-'".+%*A%1).-+ I1-'J)+/'-)+%*A%1).-+ I122L T84I !"#$%&&'#$()*# P69Q P58H I136V 234:+234F+2743+!0.75!0.50!0.250.000.2572T_wt72T_i122l72T_i38v74G_wt74G_i122l74G_i38v78H_wt78H_i122l78H_i38v82I_wt82I_i122l82I_i38v291C_wt291C_i122l291C_i38v297H_wt297H_i122l297H_i38v311C_wt311C_i122l311C_i38v320A_wt320A_i122l320A_i38v325Y_wt325Y_i122l325Y_i38v325T_wt325T_i122l325T_i38v325C_wt325C_i122l325C_i38v326Y_wt326Y_i122l326Y_i38v326H_wt326H_i122l326H_i38v336L_wt336L_i122l336L_i38v341Y_wt341Y_i122l341Y_i38vMutationfitness!"#$%&'()*+,-&-.'&$%+/-(#)/#0+ 1$*'2+34!+)00-/')#$.+ 5-%&)*'6$.+7'#($00+8$#%'/0+ 9+"9:;+"9:<;+9:<;+ !"#$%&&'#$()*# !1.6!1.4!1.2!1.0!0.8!0.672T_wt72T_i122l72T_i38v74G_wt74G_i122l74G_i38v78H_wt78H_i122l78H_i38v82I_wt82I_i122l82I_i38v291C_wt291C_i122l291C_i38v297H_wt297H_i122l297H_i38v311C_wt311C_i122l311C_i38v320A_wt320A_i122l320A_i38v325Y_wt325Y_i122l325Y_i38v325T_wt325T_i122l325T_i38v325C_wt325C_i122l325C_i38v326Y_wt326Y_i122l326Y_i38v326H_wt326H_i122l326H_i38v336L_wt336L_i122l336L_i38v341Y_wt341Y_i122l341Y_i38vMutationfitness!1.6!1.4!1.2!1.0!0.8!0.672T_wt72T_i122l72T_i38v74G_wt74G_i122l74G_i38v78H_wt78H_i122l78H_i38v82I_wt82I_i122l82I_i38v291C_wt291C_i122l291C_i38v297H_wt297H_i122l297H_i38v311C_wt311C_i122l311C_i38v320A_wt320A_i122l320A_i38v325Y_wt325Y_i122l325Y_i38v325T_wt325T_i122l325T_i38v325C_wt325C_i122l325C_i38v326Y_wt326Y_i122l326Y_i38v326H_wt326H_i122l326H_i38v336L_wt336L_i122l336L_i38v341Y_wt341Y_i122l341Y_i38vMutationfitness"=:<+"=:9+>CK=G+8A+ EAL1+ DL?@A 8B''289#:,%';'A=A>C' 289#:,%';'A=AAA@D' @AA'D>'>A'<>'A'E.0*3*.)' A'@AA''@=A'@=>'F#$*#)1%' Figure 3.4: Unique beneficial mutations have high percentages of synonymous codon fitness disparities . A. Variance of fitness metrics for synonymous codons of beneficial mutations as a fu nction of position in the primary sequence. B. Percentage of shared and unique beneficial mutations with synonymous codon fitness metric disparities. p -values reported are from contingency table analysis with 2 -tailed Fisher exact probability test. !! 57 (Figure 3.4B ). In fact, the vast majority of WT (84.2%) - and many of the I122L (42.8%) and I38V (48.6%) - unique beneficial mutations have synonymous codon fitness sign disparities (Figure 3.4B ). This indicates that transcriptional -translati onal effects impose significant evolutionary constraints. Discussion In this study we used deep mutational scanning to analyze how the probability of attaining the folded, active state impacts local fitness landscapes. AmiE I38V and AmiE I122L were desig ned and validated to have identical catalytic parameters to WT but have a lower probability of folding under selection conditions. We found that the DFE for both variants was largely similar to the AmiE WT, and that most fitness -enhancing mutations are sha red. However, there were two major surprises found when analyzing the set of mutations exhibiting positive sign epistasis. First, we expected there to be a larger subset of beneficial mutations shared only between the I122L and I38V datasets, as current m odels of stability -induced epistasis posit that many nonspecific globally -distributed mutations can improve the probability of folding 3. In contrast, we found that sign epistatic mutations were overwhelmingly specific for the I122L and I38V backgrounds. It is possible that beneficial non -specific sign epistatic mutations are the minority because of the alterations to in vivo expression that are required for the deep mutational scans. In the non -promoter tuned E. coli the impaired enzymes provide very weak c ell g rowth. It is possible that non -specific epistatic effects may have arisen to be equivalent with, or dominant to, specific epistatic phenomenon if experiments could be performed without increasing the in vivo expression of the impaired enzymes. A limitation of our deep mutational scanning experiments is ! 58 that cell growth must be proportional to enzyme function and must operate within a window of growth rate ratios 23. This requires increased in vivo expression of the impaired enzymes. By increasing t he in vivo expression it is possible that the selective advantages that the non -specific mutations might possess could be dampened. Alternatively, the protein folding pathway for homohexameric AmiE in E. coli is potentially much more complicated than model systems of monomeric, single domain proteins that have built much of the current intuition about stability and epistasis. Therefore, the sparsity of non -specific beneficial mutations could be an artifact of our experimental system. These considerations al so may explain the lack of a neutral peak for fitness expected from previous theoretical 48 and experimental 49 datasets on other proteins. More careful measurements on a wider array of oligomeric proteins should resolve this seeming contradiction. As a se cond surprise, we found that unique beneficial mutations strongly depend on codon choice, as approximately 50% of sign epistatic mutations in the I38V background show sign disparities. We speculate that this unexpected result arises from the complicated co -translational folding in vivo of the homohexameric AmiE. Local, specific nonsynonymous mutations may recover on -target folding trajectories more efficiently than nonspecific, globally stabilizing mutations. Similarly, on -pathway folding kinetics may diffe r considerably between variants, which can be selectively modulated by codon choice 50,51 . As an alternative explanation, Kudla and colleagues recently report that synonymous codons can exert fitness effects through RNA toxicity itself 52 through an unknown mechanism. While we were able to determine that both I122L and I38V mutations decrease the probability of active AmiE expression at 37¡C, we were unable to measure the relative stability of the monomeric proteins. For the thermodynamic studies, AmiE aggre gated under most ! 59 conditions, and where suitable conditions were found the lack of an isosbestic point hampered analysis. The thermal melts showed a single transition, most likely due to the hexameric dissociation. Lowering the AmiE concentration supported monomer formation at 25¡C but yielded too weak a signal for analysis of monomer thermal stability. CD melts at low AmiE concentrations did show a modest but significant decrease in Tm in the variants relative to WT. Nevertheless, our results show that simp le biophysical models currently used to model protein evolution are incomplete and that biophysical models may need to use kinetic models to account for the folding probability in vivo . Materials and m ethods Reagents All antibiotics were purchased from G oldBio and all purchased enzymes were from New England Biolabs. All other chemicals were purchased from Sigma -Aldrich. Primers and mutagenic oligos were purchased from Integrated DNA Technologies and were designed using either Benchling (www.benchling.com) or the Agilent QuikChange Primer Design Program (www.agilent.com ). Computational design of folding impaired mutants The FilterScan Rosetta script 26 was modified to predict mutations that would decrease thermodynamic stability without altering catalytic efficiency. The structural coordinates for AmiE53 (PDB: 2UXY, 341 residues per monomer) were taken from the Protein Data Bank and prepped for use in R osetta scripts through the Ôclean_pdb_keep_ligand.pyÕ script released with Rosetta 3 54. The crystal structure data was refined through the Òrefine.xmlÓ Rosetta scripts ! 60 (unaltered) from Goldenzweig et al. 26. To avoid impacting catalytic efficiency residues within 8 † of the active site were excluded from the FilterScan protocol. Additionally, surface residues were predicted and excluded from computational testing to avoid disturbing the native homohexameric state. The FilterScan script was modified to remove the input from the position specific scoring matrix (PSSM) evolutionary conservation term. Mutants with scores that predicted destabilization of the enzyme and were also shown to decrease relative fitness in a previous study 24 were selected for biophysica l analysis. Plasmid c onstruction The variants selected for biophysical analysis were constructed by mutating the AmiE WT sequence using the single mutation protocol of Nicking Mutagenesis 34. The pEDA3 constitutive expression plasmid from Wrenbeck et al. 34 was used as the vector for the mutagenesis. Variants were subcloned from the pEDA3 background into protein expression plasmid pET -29b(+) (Novagen) or into the constitutive expression plasmid pEDA2 25 at NdeI and XhoI sites using classic restriction cloni ng. Plasmid pAG was constructed by mutating the -10 and -35 promoter regions of the pEDA3 plasmid using the multi -site nicking mutagenesis protocol from Wrenbeck et al. 34. Following mutagenesis, the mutant promoter libraries were transformed into the E. co li growth selection strain MG1655 rph+ [F - #-] (Coli Genetic Stock Center #7925, CGSC strain designation: BW30270) . All AmiE variant DNA and protein sequences are listed in Supplementary Notes A 1 and 2. ! 61 Near c omprehensive single -site mutant library co nstruction Near comprehensive mutant libraries for AmiE I38V and AmiE I122L were constructed using the comprehensive nicking mutagenesis protocol from Wrenbeck et al. 34. The genes for both impaired enzymes were broken into the following tiles: tile 1 (re sidues 1 -85), tile 2 (residues 86 -170), tile 3 (residues 171 -255), and tile 4 (residues 256 -341) in pAG. For AmiE I38V 13 residues were excluded from mutagenesis (residues 32 -44), while for AmiE I122L 17 residues were excluded from mutagenesis (residues 11 5-130 and 132). Nicking mutagenesis products were amplified and purified as in Klesmith et al. 24. 10 ng each of the respective DNA libraries was transformed into E. coli MG1655 rph+ by electroporation performed with either a 1 mm electroporation cuvette at 1200 V (AmiE I122L; AmiE WT), or a 2 mm electroporation cuvette at 1600 V (AmiE I38V) using an Eppendorf Eporator. All experimental and control libraries were transformed into the selection strain with greater numbers than that required for theoretical co mplete library coverage ( Table A 6). The transformation procedure for the selection strain was optimized to minimize double plasmid transformants as described in Kowalsky et al. 23. -80¡C freezer cell stocks of the libraries were prepared as detailed in Kle smith et al. 24. Protein expression and purification pET29(b) constructs harboring genes encoding AmiE variants were transformed into E. coli BL21*(DE3) cells (Invitrogen) and expressed using Studier auto -induction 33 at 22¡C for 16 -18 hours. Cultures wer e pelleted and frozen at -80¡C. Proteins were purified from cell pellets by Ni-NTA affinity chromatography exactly as described in Klesmith et al. 24. Purified enzymes were desalted into phosphate buffered saline (PBS; 10 mM Na 2HPO4, 1.8 mM KH 2PO4, 2.7 mM ! 62 KCl, 137 mM NaCl, pH 7.4) using disposable PD -10 desalting columns (GE Healthcare), sterilized through a 0.22 µm syringe filter, and stored at 4¡C until analysis. Purified enzymes showed as a single band by SDS -PAGE ( Figure A 16). Purified enzyme solutions were quantified using the absorbance at 280 nm in 1x PBS using a published 55 theoretical A 280 molar extinction coefficient of 56,980 M -1 cm-1. For quantitative comparison of purified product yields under the T7 promoter system in BL21* E. coli , slight alt erations to the above induction scheme were used. For this analysis induction cultures were always started at an OD 600 of 0.005 from 1 mL LB + 50 µg/mL kanamycin cultures grown at 37¡C overnight. Next, 500 mL induction cultures were inoculated at an OD 600 of 0.005 with the overnight cultures and grown at 37¡C with shaking at 250x rpm for 6 hours, and following this growth step cultures were moved to 22¡C and induced for ~17 hours. Induction cultures were then pelleted and the wet cell weights of the pellets recorded. Cultures were then purified, desalted, and quantified as described above. Biophysical analysis of proteins Analysis of the growth rates of the AmiE variants in the respective constitutive expression plasmids in MG1655 rph+ E. coli were perfor med exactly as in Wrenbeck et al. 25. Assessment of the oligomeric state of the purified enzymes was performed using SEC -FPLC. Approximately 3 mL of 30 µM of the purified enzymes in PBS were run on an AKTA -FPLC system at 1 mL/min on an HiLoad 16/600 Superde x 200 column equilibrated with PBS. Enzyme kinetics (K M and k cat) was determined via phenol -alkaline hypochlorite end -point activity assays exactly as in Wrenbeck et al. 25. Kinetic analysis of purified WT and folding impaired AmiE variants was performed wi thin 6 days of purification. ! 63 To test for an isosbestic point the AmiE variants were denatured in guanidinium -HCl (GDN -HCl) and native tryptophan fluorescence was detected. Purified AmiE WT was diluted to 52 µM in increasing amounts of ice cold GDN -HCl (0 Ð 4 M) in PBS with 1 mM DTT and mixed gently. 200 µL of the denaturation mixtures were placed into opaque black 96 well plates and covered with optical film. Plates containing the samples were incubated at 4¡C for 8 hours. Next, the native tryptophan fluorescence was measured in uncovered plates using an excitation of 290nm and emission of was detected over a range of wavelengths: 310 nm Ð 370 nm. This lack of an isosbestic point indicates an unfolding model th at is more complex than a two -state model, presumably because of monomer folding and homohexamer association. Thermal shift analysis was performed exactly as described in Wrenbeck et al. 25 with incubations for 2.5 hours at 25¡C prior to addition of the d ye and initiation of the thermal shift assays; the data was processed as in Huynh et al. 56. In brief 10, 5, 2.5, 1, 0.5, and 0.25 µM purified AmiE in 1x PBS was incubated at 25¡C for 2.5 hours. Next, the samples had 5 µL of 200x SYPRO -orange dye (Life Tech nologies) added to 45 µL of the diluted enzymes in 0.1 mL MicroAmp ¨ 96-Well Reaction Plate (Life Technologies) and covered with MicroAmp ª optical film (Life Technologies). Thermal melt analysis was performed in a QuantStudio 6 Flex RT -PCR device (ThermoFis her). The melt ranged from 25¡C to 98¡C with 1¡C change per minute and with a 2 minute incubation at the first and last temperatures. Thermal melt analysis with circular dichroism, and the far -UV spectral analysis of the folded and unfolded enzymes, was performed on a Chirascan plus spectrophotometer (Applied Photophysics). Purified AmiE was buffer exchanged into 10 mM phosphate buffer pH 7.5 using PD -10 desalting columns (GE Healthcare) approximately 24 hours prior to analysis and stored at 4¡C. 1 µM sampl es were diluted in the 10 mM phosphate buffer and stored in a 25¡C water bath for at least 12 hours prior ! 64 to analysis. Prior to initiating the thermal melt analysis circular dichroism spectra were obtained from 180 nm to 260 nm at either 15¡C (10 µM sample s) or at 25¡C (1 µM samples) in a 0.5 mm cuvette with 0.5 seconds per -time-point. Thermal melt analysis of the 1 µM samples started at 25¡C and ramped to 95¡C at a rate of 1¡C/min with 0.5¡C steps and a tolerance of 0.2¡C. Signal at a wavelength of 222 nm was measured every 24 seconds throughout the melt. After the samples had reached the maximum temperature, the far -UV spectra were measured as before but with the temperature set at 95¡C for all samples. Following circular dichroism analysis the obtained sp ectra were adjusted for their respective buffer blanks and smoothed using the Savitsky -Golay filter. The Window Size of the Savitsky -Golay filter was set to 14 for smoothing all buffer blanks prior to adjusting the sample spectra, and to 6 when smoothing t he adjusted sample spectra. Finally, the obtained data was converted to Mean Residue Ellipticities (millideg * cm2 * dmol -1) prior to curve fitting. Boltzmann curve fitting was performed to determine the apparent T m for both thermal melt experiments using GraphPad Prism ( www.graphpad.com ). To assess relative activity of the refolded enzymes, enzymes were first denatured in ice cold 3 M GDN -HCl in PBS supplemented with 1 mM DTT for 16 hours at 4 ¡C at a final concen tration of 50 µM. The solution was then diluted 50 -fold into PBS with 1 mM DTT and 0.1% (w/v) BSA at 4¡C in 96 -well PCR plates that had been blocked with 1% (w/v) BSA in PBS for 1 hour at 37¡C, resulting in a total protein concentration of 1 µM. Refolding mixtures were then incubated at 4 ¡C for 5 minutes, and warmed to 37¡C over 4.5 minutes. Samples were then held at 37¡C for 20 minutes and then cooled to 4¡C and held there until assaying. Immediately prior to assaying, samples were diluted in fresh ice -col d PBS with 1 mM DTT to ensure linearity in the activity assays. Enzymes were assayed using the phenol -alkaline hypochlorite end -point assay with 20 mM acetamide as the substrate. As a control, enzymes went through the same steps ! 65 as above except without ini tial GDN-HCl denaturation. The percent activity of the refolded enzyme was determined relative to the control sample. Two biological replicates were performed for all reactions. Refolding experiments were performed within 7 days of purification of the enzy mes from the pellets. Growth selections Growth selections were performed exactly as in Wrenbeck et al. 25. Briefly, starter cultures were grown overnight in the non -selective media (M9 minimal media: 47.6 mM Na2HPO4, 22 mM KH 2PO4, 8.54 mM NaCl, 18.68 mM N H4Cl, 50 µg /mL carbenicillin, pH 7.0 ) and the following day the cells were washed in ice -cold M9 salt solution without ammonium chloride. Next, 3 mL of non -selective or selective media (M9 minimal media without ammonium chloride supplemented with 10 mM ace tamide) was inoculated with the washed cells at an initial OD600 = 0.02 (~6x10 6 cells) in Hungate tubes. Cultures were grown at 37¡C with shaking at 250x rpm for approximately 8 generations. Continuous exponential growth was ensured by harvesting cells aft er the first 4 generations and re -inoculating with 3 mL fresh media + antibiotic at OD 600 = 0.02 prior to growth for the final 4 generations. Following 8 generations of growth the cells were stored and plasmid DNA extracted as in Klesmith et al. 24. Unique selections started from the same unselected overnight culture were performed as replicates. To evaluate reproducibility between growth selections performed here and previous work performed on AmiE WT 25, a deep mutational scan of residues 171 -255 in AmiE WT was performed in parallel to each growth selection performed in the present work. ! 66 Deep mutational scanning AmiE variant DNA collected from the pre - and post -selection libraries was prepared for 300 BP paired end Illumina MiSeq sequencing as in Kowalsky et al. 23. Primers used in the PCR reactions in preparation for Illumina sequencing are listed in ( Table A 10). Sequencing of the variants was performed at the University of Illinois Chicago sequencing core. AmiE WT deep mutational scanning unprocessed sequencing results from Wrenbeck et al. 25 were downloaded from the SRA. Respective technical, and biological, replicates were processed independently. Regression analysis between the three starting points were performed by combining replicates within each variant. All data was processed using PACT 35 with the following changes from the default options entered into the configuration file: fast_filter_translate: qaverage = 20, and qlimit = 0; enrichment: ref_count_threshold = 5, sel_count_threshold = 0, strict _count_threshold = True. Normalized fitness metrics ($i) were calculated by PACT as outlined in Kowalsky et al. 23. To summarize, PACT calculates an enrichment ratio ( %i) for mutations by assessing the pre - and post -selection counts of each mutant: !!!!!!"# !!!!"!!"!! (2) Where Ä Äi is the frequency of mutant i in the post -selection population and Ä oi is the frequency in the pre -selection population. The normalized fitness metric for each mutant i ($i) was next calculated using the population -averaged number of doublings during selection (g p) and the enrichment ratios of the mutant (%i) and the unmutated starting variant (%ref ): !!!!!!"# !!!!!!!!!!!!!!!"#!!!!!!!!!! (3) Lower bound fitness me trics - the cut -off below which fitness metrics cannot be discriminated from one another - were calculated as in Wrenbeck et al. 25 by using the median ! 67 read count for the pre -selection library and other statistics produced by PACT ( Figure A 8). Lower fitness metrics were calculated to be: -1.38 for AmiE WT, -0.97 for AmiE I122L, and -0.86 for AmiE I38V. AmiE I122L had seven outlier mutations removed because the difference in fitness metrics between replicates was greater than the 99.977% confid ence intervals determined from sequencing depth of coverage ( Figure A 17). No other mutants were removed from any of the datasets. To account for global differences in fitness effects, we also generated normalized scatter plots for globally beneficial m utations. For each pairing of protein variants with AmiE WT (WT/I38V, WT/I122L), we performed a linear regression with the fitness of the first variant as the independent variable and the fitness of the second variant as the dependent variable. We then inv erted the linear transformation obtained and applied it to the second fitness. Specifically, if the fitness of the second variant Y was modeled as Y = mX + B, then the normalized fitness Y_hat was computed as (Y -B)/m. Thus, after normalization, the least -squares regression associated with each pairwise plot has a slope of one and an intercept of zero. Data a vailability Raw sequencing reads have been deposited in the Sequencing Read Archive (SRA SAMN11258744 Ð SAMN11258771 ). The processed data sets are ava ilable in Figures A 18 -20 and in Tables A 11 Ð 20. The AmiE I38V pAG and AmiE I122L pAG plasmids used in mutant library generation have been deposited in Addgene (Addgene ID: 129791 and 129792), while the AmiE WT base construct modified in Wrenbeck et al. 25 was previously deposited in Bienick et al.55 (Addgene ID: 59837). ! 68 Acknowledgements Thanks to Dr. J. Klesmith for his PACT troubleshooting help, E. Maurer and J. Hosten for their help with assorted tasks, and members of the Whitehead lab for providing feedback on ideas and figures. This work was supported by NSF CBET Career Award #1254238 to T.A.W. ! 69 REFERENCES ! 70 REFERENCES 1. 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We segmented the &X174 genome into 15 non -toxic and non -replicative fragments compatible with golden gate assembly. We next used nic king mutagenesis with oligonucleotides prepared from unamplified oligo pools with individual segments as templates to prepare near -comprehensive single -site mutagenesis libraries of genes encoding the F capsid protein (421 amino acids scanned) and G spike protein (172 amino acids scanned). Libraries possessed greater than 99% of all 11,860 programmed mutations. Golden Gate cloning was then used to assemble the complete &X174 mutant genome libraries, and libraries transformed into E. coli C were infective. T his protocol will expand reverse genetics experiments possible for studying viral evolution and, with some modifications, can be applied for engineering of therapeutically relevant bacteriophages with larger genomes. ! 76 Introduction Predicting the tempo and trajectory of evolutionary change in the complex environments encountered by viruses and bacteria remains a challenge 1-3. Understanding such changes are important for fundamental evolutionary studies as well as biotechnology applications like phage the rapy for multi -drug resistant bacteria 4. Two obstacles preventing better predictions are the oversimplification of the environment in experimental evolution studies compared to wild conditions and the vast number of possible mutational combinations that ca n occur, even in short adaptive walks and small genomes. Advances in DNA sequencing and synthesis have opened new ways to study microbial evolution and overcome some of these obstacles 5,6 . Unfortunately, suitable methods do not exist for generating compreh ensive bacteriophage mutant libraries. This technology gap hinders those seeking to engineer phage for biotechnological applications and for those seeking a deeper understanding of how viruses evolve. Methods for genetically engineering phages have recen tly been reviewed 7 and commonly involve homologous recombination and recombineering. While such approaches can be improved by clever incorporation of CRISPR -Cas systems 8-10, the overall modest efficiencies largely limits mutagenesis to the generation and s ingle -site mutation of chimeric genomes 11,12 , gene deletions 13, and limited multi -site mutagenesis 14. By contrast, human viruses often have reverse genetics systems in place where comprehensive mutant libraries can be prepared for single genes 15 or regions within a gene 16,17 using replicative plasmids that encode whole viruses or viral components. Similarly, deep mutational scanning can be performed on plasmid -encoded phage proteins like the MS2 capsid protein 18. Advances in the technologies f or generating mutant libraries, synthesizing DNA, and for assembly of large DNA fragments 19 potentially allow for the facile construction and assembly of ! 77 user -defined mutagenesis of long nucleic acids. Nicking mutagenesis (NM) can be used to construct comp rehensive single -site or other user -defined mutant libraries 20 using plasmid dsDNA as a template. In NM an oligo encodes the desired mutations by mismatch with the parental template. Recently, on -chip ink -jet printed oligo pools have been integrated into N M21, so one can now construct heterogeneous libraries of oligos that contain tens to hundreds of thousands of high fidelity, unique sequences 21,22 with a low per base pair cost. We wondered whether combining the advantages of replicative plasmids with ad vances in DNA synthesis and mutagenesis would allow us to create large user -defined libraries of bacteriophages. As a model bacteriophage system we selected &X174 because it has been extensively studied 23-26, has high resolution X -ray crystallography struc tures of the capsid and spike proteins 27,28 , and has a very small genome 29 of 5386 nucleotides. These attractive features have led to proof -of-principle demonstrations from other groups for synthetic genome assembly from oligonucleotides 30 and complete ref actoring of the phage 31. This method combines Golden Gate assembly, nicking mutagenesis, and oligo pool technology to construct near comprehensive single -site and expansive multisite mutant libraries for the genes encoding the entire capsid F protein and s pike G protein of the bacteriophage &X174 . To our knowledge this is the first time near comprehensive single site mutant libraries of full capsid and spike proteins have been generated for an infective bacteriophage. Results Our strategy for generating large user -defined mutagenesis libraries of bacteriophage &X174 is shown in Figure 4.1 . The circular 5386 nucleotide genome compactly encodes 11 genes where the F gene encodes a capsid and G encodes the spike protein ( Figure 4.1A ). The ! 78 genome was segmented onto 15 separate plasmids, including 3 plasmids for the F gene and 2 for the G gene ( Figure 4.1B ). User -defined comprehensive mutations on F and G were encoded using nicking mutagenesis with a single unamplified oligo pool containing all 11,860 mutagenic oligonucleotides ( Figure 4.1C ). &X174 mutant genomes are to be reconstituted from individual plasmids using Golden Gate cloning and transformed into a host E. coli strain ( Figure 4.1D ). Harvested phages will contain single or double non -synonymous mutations in the F and G genes depending on the mutagenized segments used for Golden Gate cloning ( Figure 4.1E ). We first sought a reverse genetics system for &X174 wherein the virus chromosome was segmented and encoded on individual plasmids. Our metho d of construction closely followed that for the assembly of human coronavirus 32 NL63, where each plasmid contained unique BsmB1 type IIS restriction endonuclease sites flanking unique five nucleotide overlaps of wild -type (WT) &X174 sequence. This architec ture allows for faithful assembly of the complete genome via Golden Gate cloning. However, as plasmids are replicated in E. coli , which &X174 naturally infects, the &X174 chromosome was encoded into15 separate nontoxic plasmids where genes were separated ! 79 !Figure 4. 1: !X174 mutant library assembly. A. Anatomy of the viral external surface. B. A linear schematic of the circular &X174 ssDNA genome. Black lines delineate portions of the virus encoded on each of the 15 replicative plasmids. C. An unamplified oligo pool, containing all user -defined mutagenic oligonucleotides for F and G genes, was created and stored as a single mixture. T his primer set was used in nicking mutagenesis for the generation of single -site saturation mutant libraries of genes encoding F and G proteins. D. Golden Gate cloning was used to assemble the mutant genomes. The dsDNA genome was transformed into E. coli and allowed a single burst phase. The resulting phage particles were collected and sequenced using SMRT sequencing. E. Linear schematics of the single site mutant libraries and some of the possible multi -site mutant libraries. *from their promoters and lar ger genes were segmented (full sequences for all plasmid inserts are given in Note B 4.1 ). &X174 phage could be reconstituted by digesting all plasmids with BsmB1, ligating inserts overnight, and transforming into electrocompetent E. coli C cells. Followin g incubation, phage plaques were tabulated. Sequencing of the genome showed that the recombinant phage encoded the intended sequence. ! 80 !The F and G genes were targeted for saturation mutagenesis. We chose to introduce mutations by nicking mutagenesis, whic h requires the presence of a unique BbvCI nicking site on the dsDNA plasmid. The F gene plasmid F3 encoding residues 246 -427 of the F gene product contained a unique BbvCI sequence, while the remaining four plasmids (F1, F2, G1, G2) required introduction o f BbvCI nicking sites in the vector backbone. The presence in each plasmid of the unique BbvCI nicking site was verified with the successful generation of circular ssDNA from the modified plasmids by BbvCI.Nt and exonuclease digestion ( Figure B 1). In nic king mutagenesis, desired mutations are encoded through libraries of mutagenic oligonucleotides, which can be sourced from unamplified oligo pools 21. We custom synthesized a single oligo pool containing 11,840 oligos encoding nearly every non -synonymous si ngle mutation in the F and G genes. Application of nicking mutagenesis using this same oligo pool for different plasmids (F1, F2, F3, G1, G2) resulted in at least 14 -fold excess transformants required for 99.9% theoretical coverage of the desired library ( Table B 1). The diversity of the mutant libraries was validated using deep sequencing on an Illumina MiSeq platform and all libraries were found to have >99% coverage of all possible single mutations (full library statistics are given in Table 4.1 ). @2<,#*ABCD*E1/2)/*,(<$2$7*F>G*"/2/("/(:"B !LVGG2;M!02=E1!KW!0.1!KW!0.1!E/=;2;/1F!X;/K;!0K! Y/;2E!Z11<0!0.1K;10/>2E!>KY1;2Z18!0.1!KY1;13!W;KG!K<1!0;2KY1;13#!!!!!!Discussion Here we present the first steps in the development of a novel method for generating comprehensive single -site saturation Ð and massive multi -site Ð virulent mutant libraries of the spike and capsid proteins of the bacteriophage &X174. This method uses unamplified oligo pools, nicking scanning mutagenesis, and Golden Gate cloning. We are fortunate that our model virus has a small genome, under 6,000 nts, which we predict will enable us to assemble the 15 Q;2ZG1<0 !Q"!Q$!Q%!U"!U$!-4!JVG=1;!KW!XKFF/=E1!GV02<0F !"8+77!$8**7!%8(&7!%8&&7!J5!T1;>1<0!0.1K;10/>2E!>KY1;2Z1 !&'!+!''!+&!J5!JVG=1;!KW!Y/2=E1! 0;213 !%&!$'!%7!(7!%7!JVG=1;!KW!-4!XE2[V1F !")!""!"'!%7!%7!! 82 non-toxic fragments with sufficient tr ansformants for near -complete coverage of the user -defined mutations. However, most biotech -relevant phages have larger genomes, and extension of this method to other viruses would encounter the following technical challenges. First, increasing genome size s decrease transformational efficiencies , and larger genomes have increased susceptibility to DNA shearing 33. Both size -dependent effects can disrupt the integrity and coverage of the phage libraries 34. Second, our method includes restrictions on the DNA sequences that can be used. Golden Gate cloning requires a genome without unique Type IIS restriction sequences, while nicking mutagenesis requires that there be only one orientation of the BbvCI nicking site within the template DNA fragment. Third, ligatin g 15 fragments is close to the upper limit of Golden Gate cloning. Additionally, increasing the size of individual fragments is difficult as the presented method depends on replicating plasmids in E. coli , and larger fragments are more likely to be toxic in vivo . Based on the above considerations, adapting this method to larger viruses will require modifications for the genome assembly steps. There are a variety of methods available for attempting to improve genome library assembly and amplification. For improving genome assembly, we speculate that a combination of hierarchical assembly 35 with other yeast based assembly methods 36 will allow for large viral genomes to be efficiently assembled. Finally, we expect the Tx.Tl cell free expression system 37 will be able to produce the viral libraries with less bias than in vivo amplification in E. coli. In summary, we have presented a complete method for efficient deep mutational scanning of the bacteriophage &X174. We anticipate this method will find utility in fundamental molecular evolution studies as well as translate to potential medicinal applications. ! 83 Materials and m ethods Reagents All purchased enzymes and DNA purification kits were from New England Biolabs, antibiotics were purchased from GoldBio, other chemicals were purchased from Sigma -Aldrich. Individual primers were purchased from Integrated DNA Technologies. Segmentation of the "X174 genome A phage assembly platform for &X174 was devised following Donaldson et al. 32. The &X174 chromosome w as divided into 15 genomic fragments designed to avoid host cell toxicity by separating genes from their promoters and breaking large genes into multiple segments ( Note B 4.1 ). Each segment is flanked by unique five nucleotide overlaps of WT &X174 sequence so that they can be amplified from the ancestral &X174 using PCR primers designed to incorporate terminal BsmB1 restriction sites. Amplicons were cloned into pCR2.1 using the Invitrogen TOPO TA cloning system (Life Technologies, Grand Island, NY). Introd uction of nicking site The gene fragments Ð F1, F2, G1, G2 Ð in the pCR2.1 -TOPO plasmid ( Notes B 4.1 and 4.2) had the BbvCI nicking site introduced via overhang PCR, type I restriction enzyme cutting, and ligation. First, PCR was performed with overhang p rimers ( Table B 2) to introduce the BbvCI site. Standard Phusion Polymerase HF reaction conditions were used with 4 ng of template DNA, cycling is follows: 98¡C - 1 min, 25x cycles of: 98¡C Ð 10 seconds, 67¡C 15 seconds, 72¡C 2.5 minutes, followed by 72¡C for 10 minutes. PCR products were run on a 1% agarose gel stained with SYBRª safe stain (Invitrogen) and the DNA bands at ~4600 bp were ! 84 extracted using a Monarch ¨ DNA Gel Extraction Kit. Next, 1 µg of the PCR product was digested with KpnI (20 U) in NEB Bu ffer 1.1 at 37¡C for two hours. Digested DNA was then clean and concentrated with a Monarch ¨ PCR and DNA Clean and Concentrate Kit and eluted into 20 µL nuclease free H 2O. One microliter of the purified and digested DNA was ligated using T4 DNA ligase at ~ 25¡C for 1 hour in standard conditions in a 20 µL reaction. Five microliters of the ligation reaction were transformed into chemically competent XL1 -Blue E. coli via standard protocols. Cells were plated on LB agar containing 100 µg/mL carbenicillin and 50 µg/mL kanamycin and grown at 37¡C for ~16 hours. Cells were picked from transformation plates and grown in 50 mL TB with 100 µg/mL carbenicillin and 50 µg/mL kanamycin for ~12 hours, cells were pelleted, and DNA purified using compact midi -preps. Compre hensive single site mutant library construction Comprehensive mutant libraries were generated using nicking mutagenesis (NM) as in Wrenbeck et al. 20 with modifications for using oligo pool mutagenic primers as noted in Medina et al. 21. A single oligo pool encoding for all possible single missense and nonsense substitutions in F and G was designed using the custom python scripts from Medina et al. 21 and custom synthesized by Agilent (full sequences of all oligos are given in the associated file ÒVMA_supplem ental_data.csv Ó in the published text). Oligo pools were designed with 20 -24 bases of gene overlap flanking the mutated codon. Codons were chosen based on E. coli codon usage frequency. This oligo pool was used directly in NM without further amplification and using 2 mg of the relevant golden gate plasmid as a template. Libraries were transformed into high efficiency electrocompetent XL1 -Blue E. coli (Agilent cat #: 200228) using 1 mm electroporation cuvettes at 1200V. Cells were plated on large bioassay pl ates (245mm x 245mm ! 85 x 25mm, Sigma -Aldrich) containing LB agar + 100 µg/mL carbenicillin and 50 µg/mL kanamycin and grown at 37¡C for ~16 hours. Plates were scraped in 10 mL plain LB, broken into ~1.2 mL aliquots, pelleted, and stored -80¡C. DNA was purifie d from 1x aliquot using a Monarch ¨ Plasmid Mini -Prep Kit. Illumina sequencing prep and analysis Purified library DNA was prepared for deep sequencing as in Kowalsky et al. 38 using the primers listed in Table B 3 and gene tiling as specified in Table 4.1 . DNA was Illumina sequenced on a Mi -Seq platform with 250 BP paired end reads. The University of Colorado BioFrontiers Sequencing Core performed the Illumina sequencing. Data was processed using PACT 39 to determine the library coverage with the following changes to the default options in the configuration file: fast_filter_translate: qaverage = 20, qlimit = 0; enrichment: ref_count_threshold = 5, sel_count_threshold = 0, strict_count_threshold = True. The heatmaps of counts can be found in Figu re B 2 - 9. Assembly of mutant genomes We pooled plasmid DNA containing all 15 of the phage DNA fragments in equimolar amounts and digested them with BsmB1 (Fermentas Fast Digest, Life Technologies, Grand Island, NY) for 30 minutes to 1 hour at 37¡C. The digested plasmids were subjected to agarose gel electrophoresis for 10 to 15 minutes using a 1.2% agarose gel to separate the vector from the inserts. The inserts were excised from the gel, purified using the GeneJET gel extraction kit (Fermentas), ligated overnigh t at 14¡C with T4 DNA ligase (Promega Corporation, Madison, WI), and transformed by electroporation into 100 µl competent E. coli C cells. The ! 86 transformation mix was resuspended with 1 ml of (LB and either plated immediately or incubated for about 20 minut es at 37¡C to allow for one viral burst. The (LB was added to 3 ml of &LB top agar and plated onto a &LB agar plate. After four to five hours of incubation at 37¡C, recombinant phage plaques were visible and plates were removed from the incubator. To verif y that the recombinant phage encoded the intended sequence, we picked about 30 plaques for each intended mutational target and Sanger sequenced the entire targeted gene. We also sequenced the F and G genes for about 30 wild type plaques to assure that no m utations were naturally accumulating. Briefly, individual plaques were picked with sterile toothpicks and placed in 200 uL &LB and gently swirled. 1 uL of this mix was used to PCR amplify approximately ) of the &X174 genome using &X-0F (5Õ -GAGTTTTATCGCTTCC ATG-3Õ) and &X-2953R (5Õ -CCGCCAGCAATAGCACC -3Õ) primers. Internal sequencing primers &X-979F (5Õ -CGGCCCCTTACTTGAGG -3Õ) and &X-1500R (5Õ -TTGAGATGGCAGCAACGG -3Õ) were used to sequence gene F. &X-2953R was used to sequence gene G. PCR cleanups and sequencing w as done at Eurofins Genomics. PCR reaction conditions were; 5 uL10X Taq buffer, 2.5 uL 10 uM &X-0F primer, 10 uM &X-2953F primer, 0.8 uL 12.5 uM dNTPs, 0.5 uL Taq polymerase (NEB #M0273), 1 uL template, 37.7 uL H 20. Thermocycling conditions were 1 cycle at 95¡C for 2 min, 30 cycles at 95¡C for 15 sec, 52¡C for 30 sec, 68¡C for 2 min, 1 cycle at 68¡C for 5min. ! 87 REFERENCES ! 88 REFERENCES 1. Holmes EC: What can we predict about viral evolution and emergence? . Curr Opin in Vir 2013, 3:180 -184. 2. 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Nature 1992, 355:137 -143. ! 90 28. Dokland T, McKenna R, Ilag LL, Bowman BR, Incardona NL, Fane BA, Rossmann MG: Struc ture of a viral procapsid with molecular scaffolding . Nature 1997, 389:308 -313. 29. Sanger F, Coulson AR, Friedmann T, Air GM, Barrell BG, Brown NL, Fiddes JC, Hutchinson CA 3 rd, Slocombe PM, Smith M: The nucleotide sequence of bacteriophage phiX174 . J Mol Biol 1978, 125:225 -246. 30. Smith HO, Hutchinson III CA, Pfannkoch C, Venter JC: Generating a synthetic genome by whole genome assembly: !X174 bacteriophage from synthetic oligonucleotides . Proc. Natl. Aca. Sci. USA 2003, 100:15440 -15445. 31. Jaschke PR, Lieberman EK, Rodriguez J, Sierra A, Endy D: A fully decompressed synthetic bacteriophage !X174 genome assembled and archived in yeast . Virology 2012, 434:278 -284. 32. Donaldson EF, Yount B, Sims AC, Burkett S, Pickles RJ, Baric RS: Systematic Asse mbly of a Full -Length Infectious Clone of Human Coronavirus NL63 . J. Virol. 2008, 82:11948 Ð11957. 33. Rogers SO, Bendich AJ: Extraction of DNA from plant tissues . Plant Mole Biol Manual 1988, A6:1 -10. 34. Edwards RA, Rohwer F: Viral metagenomics . Nature Reviews Micro 2005, 3:504 -509. 35. Lee, ME, DeLoache WC, Cervantes B, Dueber JE: A highly characterized yeast toolkit for modular, multipart assembly . ACS Synth Biol 2015, 4:975 -986. 36. Ando H, Lemire S, Pires DP, Lu TK: Engineering modular viral s caffolds for targeted bacterial population editing . Cell Systems 2015, 1:187 -196. 37. Garamella J, Marshall R, Rustad M, Noireaux V: The all E. coli Toolbox 2.0: a platform for cell -free synthetic biology . ACS Synth Biol 2016, 5:344 -355. 38. Kowalsky CA, Klesmith, JR, Stapleton JA, Kelly V, Reichkitzer N, Whitehead TA: High -Resolution Sequence -Function Mapping of Full -Length Proteins . PLoS One 2015, 10:e0118193. 39. Klesmith JR, Hackel BJ: Improved mutation function prediction via PACT: Protein Analysis an d Classifier Toolkit . Bioinformatics 2018, doi/10.1093/bioinformatics/bty1042. !!! ! 91 CHAPTER 5 Conclusions and p erspectives ! 92 This thesis focuses on the generation and analysis of large mutational datasets for understanding molecular evolution. Deep mutational scanning (DMS) is a technique that combines saturation mutagenesis libraries with high throughput selection and deep sequencing to assess the impact of nearly all single -point mutations on protein function 1. Here, DMS is used to study how folding probability constrains the molecular evolution of enzymes. This thesis also expands the research questions DMS experiments can address through a novel method for generating mutant genome libraries of virulent phages. This chapter provides a summary of the work performed and a discussion of the broader impacts of this PhD thesis. DMS experiments produce large datasets that quantitatively describe the impacts of single mutations 2-5. In Chapter 2 , we reviewed the use of DMS datasets - as we ll as datasets from several other experimental and evolutionary sources Ð in the forward engineering of protein therapeutics. We describe the use of these datasets in the identification of mutations that can tune the specificity, affinity, and solubility o f a target protein. As more comprehensive studies of mutational impacts are performed, we expect the integration of the resultant data to provide better predictions of which mutations will, and will -not, work toward a specific goal. Eventually, we expect g uidelines like LipinskiÕs rule of five for small molecule drugs 6 to emerge for protein therapeutics. Indeed, work aiming to establish these guidelines are already underway 7. In Chapter 3 , I demonstrated how DMS can be used to understand how a single biop hysical parameter of an enzyme constrains its molecular evolution. In this study I performed near -comprehensive single -mutation analysis on two unique single -point mutants of the aliphatic amidase AmiE from P. aeruginosa . The point -mutants have decreased in vivo folding probabilities and statistically indistinguishable catalytic activities compared to the starting enzyme. In a previous study 5 the starting enzyme had undergone DMS with the same selection ! 93 conditions applied in this study, allowing us to compa re the obtained datasets. Because the only biophysical parameter modulated is the in vivo folding probability, we are able to assess how only this parameter alters the types of mutations that can arise. Additionally, this study provides a near comprehensiv e analysis of how single -point mutations combine, providing a detailed view of epistasis, the non -additive combination of mutational effects 8. The novelty of this study is found in the aforementioned details and in the fact that this study is the most comp rehensive analysis of how a single initial biophysical parameter impacts mutational outcomes to date. Comparing the datasets obtained for our two disrupted enzymes with that of the initial starting enzyme provides intriguing insights into molecular evolu tion. First, in all genetic backgrounds the distributions of beneficial fitness effects are described by the General Pareto distribution. Thus, the likelihood of uncovering a beneficial mutation is insensitive to initial in vivo folding probability in our experiments. Interestingly, most beneficial mutations are shared, and only 19 mutations are likely to require an increased in vivo folding probability to buffer stability penalties. Epistasis was found in both the beneficial and deleterious populations o f mutations following decreases in the in vivo folding probabilities. Interestingly, in the population of double mutants that are deleterious individually and beneficial when combined, phenotype -specific interactions dominated those that are phenotype -independent. This suggests that mutations rescuing in vivo folding probability are more likely to be in direct contact with the disrupting mutation, and less likely to alter the global properties of the enzyme. Additionally, beneficial mutations with synonymous codon fitness disparities - opposite mutation al outcomes for synonymous codons - were correlated with background specific beneficial mutations. This ! 94 correlation reveals the importance of the mRNA sequence selected, and how it significantly restricts mutational outcomes for identical peptides. Taken together, these results indicate that models of protein stability and evolution must include all aspects of the protein life cycle, from transcription to degradation, to fully understand the impact of a single mutation. It is uncertain if these findings a re unique to this experimental system, or if they are descriptive of protein evolution in general. Additional studies similar to this one, using other proteins and with different biophysical parameters altered, will improve our ability to predict mutationa l outcomes. The final goal of this thesis was methodological. In Chapter 4 we expanded the experimental range of DMS by generating comprehensive single -site saturation, and massive multi -site, mutant genome libraries of the bacteriophage 'X174. The mutan t genomes were generated by performing nicking scanning mutagenesis 9 on non-toxic truncations of the genome and assembling complete viral genomes with Golden Gate cloning. These mutant genomes will allow researchers to probe how host specificity is encoded into the primary sequence, how intra - and inter -molecular epistasis is involved in viral evolution and micro -compartment formation, and expand our understanding of the molecular evolution of viruses. In conclusion, this thesis provides significant insig hts into molecular evolution and how to study it. All aspects of this thesis involve the generation and/or analysis of large mutational datasets with the goal of understanding how proteins change over evolutionary time. In the coming decades the mutational data obtained from more DMS experiments, and from other experimental and archival sources, will provide valuable insights into molecular evolution. I expect this information will be combined and integrated into a new generation of predictive algorithms, w hich will significantly improve the protein engineering process. ! 95 REFERENCES ! 96 REFERENCES 1. Fowler DM, Fields S: Deep mutational scanning: a new style of protein science. Nat Methods 2014, 11:801 Ð807. 2. Kowalsky CA, Klesmith JR, Stapleton JA, Kelly V, Reichkitzer N, Whitehead TA: High -Resolution Sequence -Function Mapping of Full -Length Proteins . PLoS One 2015, 10:e0118193. 3. Klesmith JR, Bacik JP, Michalczyk R, Whitehead TA: Comprehensive Sequence -Flux Mapping of a Levoglucosan Utilization Pathway in E. coli. ACS Synth Biol 2015, 4:1235 Ð1243. 4. Klesmith JR, Bacik JP, Wrenbeck EE, Michalczyk R, Whitehead TA: Trade -offs between enzyme fitness and solubility illuminated by deep mutational scanning . Proc Natl Acad Sci U S A 2017, 114:2265 -2270. 5. Wrenbeck EE, Azouz LR, Whitehead TA: Single -mutation fitness landscapes for an enzyme on multiple substrates reveal specificity is globally encoded . Nat Comm 2017, 8:15695. 6. Lipinski CA: Lead -and drug -like compounds: the rule -of-five revolution . Drug Discov Today Technol 2004, 1:337 -341. 7. Rabia LA, Desai AA, Jhajj HS, Tessier PM: Understanding and overcoming trade -offs between antibody affinity, specificity, stability and solubility . Biochem Eng J 2018, 137:365 -374. 8. Starr TN, Thornton JW: Epistasis in protein evolution . Protein Science 2016, 25:1204 -1218. 9. Wrenbeck EE, Klesmith JR, Stapleton JA, Adeniran A, Tyo KEJ, Whitehead TA: Plasmid -based one -pot saturation mutagenesis . Nat Methods 2016, 13:928 -930. !!!!!!!!!!!! 97 !! APPENDICES ! 98 APPENDIX A Chapter 3 supporting information ! 99 Note A 1: Amino acid sequences of the AmiE variants. Mutations are highlighted in red. >AmiE_WT MRHGDISSSNDTVGVAVVNYKMPRLHTAAEVLDNARKIAEMIVGMKQGLPGMDLVVF PEYSLQGIMYDPAEMMETAVAIPGEETEIFSRACRKANVWGVFSLTGERHEEHPRKAPY NTLVLIDNNGEIVQKYRKIIPWCPIEGWYPGGQTYVSEGPKGMKISLIICDDGNYPEIWRD CAMKGAELIVRCQGYMYPAKDQQVMMAKAMAWANNCYVAVANAAGFDGVYSYFG HSAIIGFDGRTLGECGEEEMGIQYA QLSLSQIRDARANDQSQNHLFKILHRGYSGLQASG DGDRGLAECPFEFYRTWVTDAEKARENVERLTRSTTGVAQCPVGRLPYEGLEHHHHHH >AmiE_I122L MRHGDISSSNDTVGVAVVNYKMPRLHTAAEVLDNARKIAEMIVGMKQGLPGMDLVVF PEYSLQGIMYDPAEMMETAVAIPGEETEIFSRACRKANVWGVFSLTGERHEEHPRKAPY NTLVLLDNNGEIVQKYRKIIPWCPIEGWYPGG QTYVSEGPKGMKISLIICDDGNYPEIWR DCAMKGAELIVRCQGYMYPAKDQQVMMAKAMAWANNCYVAVANAAGFDGVYSYF GHSAIIGFDGRTLGECGEEEMGIQYAQLSLSQIRDARANDQSQNHLFKILHRGYSGLQAS GDGDRGLAECPFEFYRTWVTDAEKARENVERLTRSTTGVAQCPVGRLPYEGLEHHHHH H >AmiE_I38V MRHGDISSSNDTVGVAVVNYKMPRLHTAAEVLDNARK VAEMIVGMKQGLPGMDLVV FPEYSLQGIMYDPAEMMETAVAIPGEETEIFSRACRKANVWGVFSLTGERHEEHPRKAP YNTLVLIDNNGEIVQKYRKIIPWCPIEGWYPGGQTYVSEGPKGMKISLIICDDGNYPEIWR DCAMKGAELIVRCQGYMYPAKDQQVMMAKAMAWANNCYVAVANAAGFDGVYSYF GHSAIIGFDGRTLGECGEEEMGIQYAQLSLSQIRDARANDQSQNHLFKILHRGYSGLQAS GDGDRGLAECPFEFYRTWVTDAEKARENVERLTRSTTGVAQCPVGRLPYEGLEHHHHH H ! 100 Note A 2: DNA sequences of the AmiE variants. Codons mutated are underlined and bold, while mutations are highlighted in red. >AmiE_WT !!ATGAGACATGGCGATATTAGCTCGTCAAATGATACCGTAGGCGTAGCCGTGGTG AATTACAAGATGCCGCGTTTACATACTGCTGCTGAAGTCCTGGATAATGCCCGCAAAAT TGCGGAAATGATCGTTGGTATGAAGCAAGGTCTGCCGGGCATGGATCTGGTTGTGTT TCCTGAATATTCTTTACAGGGTATTATGTACGACCCTGCTGAAATGATGGAAACAGC CGTGGCGATTCCAGGCGAAGAAACGGAAATCTTTAGCCGTGCTTGTAGAAAAGCAA ATGTTTGGGGTGTGTTCTCCCTGACCG GCGAACGTCATGAAGAACACCCTAGAAAGG CACCATACAACACTCTGGTCTTGATCGATAACAACGGTGAAATCGTACAAAAGTAC AGAAAGATCATCCCATGGTGTCCGATTGAAGGCTGGTATCCAGGTGGCCAGACATA CGTCTCTGAAGGTCCGAAAGGCATGAAGATCTCATTAATTATCTGCGATGACGGTAA TTATCCGGAAATTTGGAGAGATTGTGCCATGAAGGGTGCGGAATTGATCGTTCGCTG CCAAGGCTATATGTACCCTGCTAAAGACCAACAAGTTATGATGGCTAAGGCAATGG CCTGGGCGAATAACTGTTATGTCGCTGTAGCAAACGCTGCAGGTTTTGATGGCGTTT ATAGCTACTTCGGTCATAGTGCCATTATCGGTTTTGACGGCCGTACTCTGGGTGAAT GCGGCGAAGAAGAAATGGGCATTCAATACGCGCAGTTGTCTCTGTCACAAATCCGC GATGCCCGTGCGAATGACCAAAGTCAGAAC CATTTGTTTAAAATCTTGCACAGAGGT TACTCCGGTTTGCAGGCTTCGGGCGATGGCGACCGTGGTCTGGCAGAATGCCCATTT GAATTCTACCGTACCTGGGTTACTGATGCTGAAAAGGCAAGAGAAAACGTGGAACG CCTGACTCGCTCCACAACAGGTGTCGCCCAATGCCCAGTAGGTCGTCTGCCGTATGA AGGCCTCGAGCACCACCACCACCACCAC >AmiE_I122L ATGAGACATGGCGATA TTAGCTCGTCAAATGATACCGTAGGCGTAGCCGTGGTGAA TTACAAGATGCCGCGTTTACATACTGCTGCTGAAGTCCTGGATAATGCCCGCAAAAT TGCGGAAATGATCGTTGGTATGAAGCAAGGTCTGCCGGGCATGGATCTGGTTGTGTT TCCTGAATATTCTTTACAGGGTATTATGTACGACCCTGCTGAAATGATGGAAACAGC CGTGGCGATTCCAGGCGAAGAAACGGAAATCTTTAGCCGTGCTTG TAGAAAAGCAA ATGTTTGGGGTGTGTTCTCCCTGACCGGCGAACGTCATGAAGAACACCCTAGAAAGG CACCATACAACACTCTGGTCTTG CTCGATAACAACGGTGAAATCGTACAAAAGTAC AGAAAGATCATCCCATGGTGTCCGATTGAAGGCTGGTATCCAGGTGGCCAGACATA CGTCTCTGAAGGTCCGAAAGGCATGAAGATCTCATTAATTATCTGCGATGACGGTAA TTATCCGGAAATTTGGAGA GATTGTGCCATGAAGGGTGCGGAATTGATCGTTCGCTG CCAAGGCTATATGTACCCTGCTAAAGACCAACAAGTTATGATGGCTAAGGCAATGG CCTGGGCGAATAACTGTTATGTCGCTGTAGCAAACGCTGCAGGTTTTGATGGCGTTT ATAGCTACTTCGGTCATAGTGCCATTATCGGTTTTGACGGCCGTACTCTGGGTGAAT GCGGCGAAGAAGAAATGGGCATTCAATACGCGCAGTTGTCTCTGTCAC AAATCCGC GATGCCCGTGCGAATGACCAAAGTCAGAACCATTTGTTTAAAATCTTGCACAGAGGT TACTCCGGTTTGCAGGCTTCGGGCGATGGCGACCGTGGTCTGGCAGAATGCCCATTT GAATTCTACCGTACCTGGGTTACTGATGCTGAAAAGGCAAGAGAAAACGTGGAACG ! 101 CCTGACTCGCTCCACAACAGGTGTCGCCCAATGCCCAGTAGGTCGTCTGCCGTATGA AGGCCTCGAGCACCACCACCA CCACCAC >AmiE_I38V ATGAGACATGGCGATATTAGCTCGTCAAATGATACCGTAGGCGTAGCCGTGGTGAA TTACAAGATGCCGCGTTTACATACTGCTGCTGAAGTCCTGGATAATGCCCGCAAA GTTGCGGAAATGATCGTTGGTATGAAGCAAGGTCTGCCGGGCATGGATCTGGTTGTGTT TCCTGAATATTCTTTACAGGGTATTATGTACGACCCTGCTGAAATGATGGAAACAGC CGTGGCG ATTCCAGGCGAAGAAACGGAAATCTTTAGCCGTGCTTGTAGAAAAGCAA ATGTTTGGGGTGTGTTCTCCCTGACCGGCGAACGTCATGAAGAACACCCTAGAAAGG CACCATACAACACTCTGGTCTTGATCGATAACAACGGTGAAATCGTACAAAAGTAC AGAAAGATCATCCCATGGTGTCCGATTGAAGGCTGGTATCCAGGTGGCCAGACATA CGTCTCTGAAGGTCCGAAAGGCATGAAGATCTCATTAA TTATCTGCGATGACGGTAA TTATCCGGAAATTTGGAGAGATTGTGCCATGAAGGGTGCGGAATTGATCGTTCGCTG CCAAGGCTATATGTACCCTGCTAAAGACCAACAAGTTATGATGGCTAAGGCAATGG CCTGGGCGAATAACTGTTATGTCGCTGTAGCAAACGCTGCAGGTTTTGATGGCGTTT ATAGCTACTTCGGTCATAGTGCCATTATCGGTTTTGACGGCCGTACTCTGGGTGAAT GCGGCGAAGA AGAAATGGGCATTCAATACGCGCAGTTGTCTCTGTCACAAATCCGC GATGCCCGTGCGAATGACCAAAGTCAGAACCATTTGTTTAAAATCTTGCACAGAGGT TACTCCGGTTTGCAGGCTTCGGGCGATGGCGACCGTGGTCTGGCAGAATGCCCATTT GAATTCTACCGTACCTGGGTTACTGATGCTGAAAAGGCAAGAGAAAACGTGGAACG CCTGACTCGCTCCACAACAGGTGTCGCCCAATGCCCAGTA GGTCGTCTGCCGTATGA AGGCCTCGAGCACCACCACCACCACCAC ! 102 Figure A 1: Chromatogram of purified AmiE variants. SEC-FPLC chromatograms of AmiE proteins run on a HiLoad 16/600 Superdex 200 column at 1 mL/min in PBS as the mobile phase. No gross differences in oligomeric state were determined for the proteins. !"#!"!!#$%!"!!#%!!"!!#&%!"!!#'!!!"!!#'$%!"!!#'%!!"!!#'&%!"!!#$!!!"!!#$$%!"!!#(!"!!#(%"!!#%!"!!#%%"!!#)!"!!#)%"!!#&!"!!#&%"!!#*!"!!#*%"!!#+!"!!#,-.#/0,12# 3415678#97410:#/0;2# <=#>?*9# >'$$;#! 103 Figure A 2: Representative far -UV spectra of the folded and unfolded AmiE variants. Far-UV spectra of folded and unfolded 10 µM AmiE WT, 10 µM AmiE I122L, 10 µM AmiE I38V at 15¡C (folded) and 90¡C (unfolded) in 10 mM phosphate buffer at pH 7.5. !"#$%&!'$%&!#$%&#$%& '$%& "#$%& "'$%& "(%&")%&#*%&#"%&##%&#+%&#,%&#%%&!"#$%&"'()*"%"++(,-(.(-/%0)"1%.234%25+3678%97:::% ;#<"+"$1-=%0$28% -./0&12&345676& -./0&8"##9&345676& -./0&8+(:&345676& -./0&12&;<345676& -./0&8"##9&;<345676& -./0&8+(:&;<345676& !"#$%$&'()*+&,-* .$"&*/$0123$* $%%14(151(6 *++2$' *5,7*,8%9:-*;:<<<-*! 104 Figure A 3: Locations of I38V and I122L mutations in AmiE quaternary structure . A. Three perspectives on the location of the disrupting I38V mutation, the residue I38 is shown as orange spheres. B. Three perspectives on the location of the disrupting I122L mutation, the residue I122 is shown as green spheres. Both mutations are in the monomer core and are not be predicted to impact quaternary structure or assembly. !"#$!"#$!"#$!"#$%&$$ '&$$ ! 105 Figure A 4: AmiE WT tryptophan emission spectra as a function of GDN -HCl concentration . 52 µM Protein was incubated in PBS with the indicated concentration of GDN -HCl at 4o C for 8 hr before fluorescence measurements. The excitation wavelength was 290 nm, while emission was detected at 315 -370 nm. The lack of an isosbestic point between spectra indicates more complicated unfolding than a two -state model. !"#$!"$!!"%$!"&!!!"&#$!"&$!!"&%$!"#!!!"##$!"#$!!"#%$!"'!!!"'#$!"'$!!"'%$!"'&$"'#!"'#$"''!"''$"'(!"'($"'$!"'$$"')!"')$"'%!"!"#$%&'$()*$+,-$ .,/00/1+$234&5&+678$%+,-$ !"*"+,-./01" #"*"+,-./01" '"*"+,-./01" ("*"+,-./01" ! 106 Figure A 5: Thermal shift analysis of the purified AmiE variants. A. Melt curves for 10, 5, and 1 M AmiE WT. B. Melt curves for 10, 5, and 1 M AmiE I122L. C. Melt curves for 10, 5, and 1 M AmiE I38V. All experiments were performed with biological (n=2) and techn ical (n=3) replicates, and representative curves are shown. !"#!!$!!!"%!!$!!!"&!!$!!!"'!!$!!!"($!!!$!!!"($#!!$!!!"($%!!$!!!")*"&#"&*"*#"**"+,-./0120320" 40560/78-/0"9:;" <5=>"?4"(!"@A" <5=>"?4")"@A" <5=>"?4"("@A" !"#!!$!!!"%!!$!!!"&!!$!!!"'!!$!!!"($!!!$!!!"($#!!$!!!"($%!!$!!!")*"&#"&*"*#"**"+,-./0120320" 40560/78-/0"9:;" <5=>"BC'D"(!"@A" <5=>"BC'D")"@A" <5=>"BC'D"("@A" !"#!!$!!!"%!!$!!!"&!!$!!!"'!!$!!!"($!!!$!!!"($#!!$!!!")*"&#"&*"*#"**"+,-./0120320" 40560/78-/0"9:;" <5=>"B(##E"(!"@A" <5=>"B(##E")"@A" <5=>"B(##E"("@A" !"## $"## %"##! 107 Figure A 6: Activity loss of AmiE WT following dilution. Comparison of the relative specific AmiE reaction velocity in a saturating amount of 20 mM acetamide following incubations at dil ute concentration in 1x PBS over a time course. Dotted and solid lines represent the best fit linear regressions for the respective data sets (n=6). !"#$!"##$!"%$!"%#$!"&$!"&#$!"'$!"'#$!"($!"(#$)$!$*$+$%$'$)!$,-./012-$34-51615$7-.85109$ :1;-$<=8>?@A$ #!!$BC$D;1E$F:$ (!!$BC$D;1E$F:$ ! 108 Figure A 7: Thermal denaturation monitored by far -UV circular dichroism. Melt curves for AmiE WT (blue), AmiE I122L (green), and AmiE I38V (orange). All experiments were performed by scanning !222nm at 1 µM protein concentration with 2 biological replicates (different colors on each panel represent a replicate experiment). Rela tive ellipticity ranges from completely folded state (0) to unfolded (1). 50607080900.00.51.0Data 1LegendLegend50607080900.00.51.0Copy of Copy of Data 1LegendLegend50607080900.00.51.0Copy of Data 1tempLegendLegend50 60 70 80 90 Temperature (¡C) 0 0.5 1.0 0 0.5 1.0 0 0.5 1.0 Relative Ellipticity AmiE WT AmiE I122L AmiE I38V ! 109 Figure A 8: Frequency distribution of pre -selection read counts for AmiE libraries. Mean and median pre -selection read counts for AmiE variants are reported on the plot s. !"#!!"$!!"%!!"&!!"'!!"(!!")!!"*!!"#"#!"#!!"#!!!"#!!!!"!"#$%#&'()*+,-.)/0123) !"#!!"$!!"%!!"&!!"'!!"(!!")!!"*!!"#"#!"#!!"#!!!"#!!!!"!"#$%#&'()*+,-.)/45563) !"#!!"$!!"%!!"&!!"'!!"(!!")!!"*!!"#"#!"#!!"#!!!"#!!!!"!"#$%#&'()*+,-.3) Mean 161.5 Median 115 Mean 155.6 Median 100 Mean 300.8 Median 169 +,-./-0-12345",-67"14852/" ! 110 Figure A 9: Deep mutational scanning replicates for AmiE WT compared with previous literature. Comparison of relative fitness metrics for a AmiE WT mutational library covering residues 171 -255 to those from same mutants reported originally in Wrenbeck et al (2017). These replicate deep mutational scans were performed in parallel to AmiE variant grow th selections as internal controls. PearsonÕs correlation coefficients reported on plots. A. control for AmiE I122L growth selection. B. control for AmiE I38V growth selection. !1.0!0.50.00.5!1.0!0.50.00.5WT tile 3 Exp combinedWT tile 3 ctrl I38V !1.0!0.50.00.5!1.0!0.50.00.5WT tile 3 Exp combinedWT tile 3 ctrl i122l bio rep 2 !"#"$%&'"" !"#"$%&$"" !()*+,-(".,+/(00"1234" 52(/6(78"(+"*)%"9'$:;<"" !()*+,-(".,+/(00"!(=),7*+(":" !()*+,-(".,+/(00"1234" 52(/6(78"(+"*)%"9'$:;<"" >:%$">$%?"$%$" $%?" >:%$">$%?"$%$" $%?" !()*+,-(".,+/(00"!(=),7*+("'" >:%$">$%?"$%$" $%?" >:%$">$%?"$%$" $%?" @%" A%" ! 111 Figure A 10: Relative fitness metrics for AmiE proteins as a function of pre-selection read counts . Absolute PearsonÕs correlation coefficients reported on plots are below 0.04 in all cases, showing that less than 0.2% of the variance can be explained by initial frequency of a given mutant in the library. !1.0!0.50.00.50500100015002000WT_pre_sel_countsWT_fitness!"#$%&&'()*"+,' -'-./' 0-./'-'/--'1---'1/--'2---'3'4'-.-11' !1.0!0.50.00.5010002000300040005000I122l_pre_sel_countsI122L_fitness!"#$%&&'()*"+'51226,' -'-./' 0-./'01'-'1---'2---'7---'8---'/---'3'4'-.-29' 01'!1.0!0.50.00.51.005001000I38V_pre_sel_countsI38V_fitness!"#$%&&'()*"+'57:;,' -'-./' -'/--'1---'1'3'4'0-.-7<9' 0-./'01'=>%0?%@%A#"B$'3%CD'EBF$#&' ! 112 Figure A 11: Normalized distribution of fitness effects for WT, I122L, and I38V backgrounds. 0.000.020.040.06!1.5!1.0!0.50.00.51.0BinWt_freq0.30.40.5!1.5!1.0!0.50.00.51.0BinWt_freq!"#$"#%#$#&#"'"# ()'!#()'"#("'!#("'"#"'!# )'"# ! !"#$"%&'&(&)*'+,&)&-(%.'-%'/-%' ! 113 Figure A 12: Analysis of proportions of deleterious mutations. Cumulative distribution functions for the deleterious mutations for each enzyme variant are plotted above the lower bounds of experimental measurements. !"#!!"$!!!"$#!!"%!!!"%#!!"&!'(" &!')" &!'*" &!'+" &!'#" &!'," &!'-" &!'%" &!'$" !"./010234" 53671829:;"<204:=="7:062>" ?72@" AB" ?72@" C$%%D"?72@" C-)E"! 114 Figure A 13: Venn diagram of the shared and unique beneficial mutations using the strict cutoff. Mutations had to improve the growth rate by "10% to be classified as a beneficial mutation ($i " 0.138). !"#$%&'# $%&'#()*+# $%&'#(,--.# )#/#,#01#0#-2#-#! 115 Figure A 14: Correlation analysis of linear regression normalized shared beneficial mutations. Comparison of the linear regression nor malized fitness metrics for the beneficial mutations shared by all enzymes; solid lines represent the linear function Y = 1X + 0. A. Comparison of linear regression normalized AmiE I38V fitness metrics with AmiE WT. B. Comparison of linear regression norma lized AmiE I122L fitness metrics with AmiE WT. !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!0.000.250.500.000.250.50WT fitnessNormalized I38V fitness !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!0.000.250.500.000.250.50WT fitnessNormalized I122L fitness !"## $"## ! 116 Figure A 15: Dot plots of fitness effect synonymous codon variances for beneficial mutations. 5#! :K0!XEK0F!KW!0.1!W/0<1FF!1WW1>0!FMK3K1F!WK;!1/0.1;!0.1! =1<1W/>/2E!GV020/K2013!//2E!GV020/KK1!/<01;Y2E!>V0KWWF^#!4.1!3/F0;/=V0/K1F!/F! ;1X;1F1<013!=M!0.1!Y/KE/KEK;13!G2;?1;!E/<1F!;1X;1F1<0!0.1!G121!WK;!0.1!XKXVE20/K<8!/2<0EM! ./Z.!Y2;/2<>1!0.20! FMK3K1F!WK;!0.1!F.2;13!2<3!V/2E!GV020/K0/Y1!1K1!/<01;Y2E!>V0KWWF^!4.1!3/F0;/=V0 /K1F!/F! ;1X;1F1<013!=M!0.1!Y/KE/KEK;13!G2;?1;!E/<1F!;1X;1F1<0!0.1!G121!WK;!0.1!XKXVE20/K<#!S%*c!.2F!2!F/Z2<0EM!./Z.!Y2;/2<>1!//2E! GV020/K&'+3$ ,-$ 4)(?4+$ 51667$$ />&'+3$ 51667$ 4)(?4+$ 59:%$ />&'+3$ 59:%$ 4)(?4+$ @#$ 1#22$ 2#221$ 1;12<=$ABC-DE+$F.$G+)+.(*(&H$I40&0(F)$ ! 117 Figure A 16: SDS -PAGE analysis of the purity of the purified AmiE variants. Samples were denatured in SDS -PAGE loading buffer (LaemmliÕs buffer supplemented to 1.5% *-mercaptoethanol at 1x) at 98¡ for 10 minutes. Samples run on 4 -20% Mini -PROTEAN ¨ TGXª precast gels (Bio -Rad) at 120 V for 1 hour and were washed and stained with SimplyBlue SafeStain (Thermo -Fisher) as described by the manufacturer. Molecular weight ruler used is the PageRuler Prestained Protein Ladder (10 -180 kDa, Thermo -Fisher). !"#$%&'$ !(#$%&'$ !)#$%&'$ !)*$%&'$ !+*$%&'$ !##$%&'$ !,*$%&'$ !)**$%&'$ !)"*$%&'$ !),*$%&'$ -'./012/3$$ 45$6'77/3$ 89:;$5<$ 89:;$=">?$ 89:;$=)((6$ (@,$A.$ *@(,$A.$ (@,$A.$ *@(,$A.$ (@,$A.$ *@(,$A.$ ! 118 Figure A 17: Comparison of AmiE I122L outlier mutation technical replicates. Mutations removed from analysis have their fitness metrics for the respective technical replicates shown, error bars represent the 99.977% confidence intervals (3.5x +) for the fitness metrics. This indicates that errors such as these should occur less than once per dataset for the size of our experiment. !"#$%&'()*+,'-.)//+0)-#'1+ 02-%-'".+ 34566+37566+38566+39566+3:566+6566+ :566+ ;$'<+=:99>+?)@&'1%-)+:+ ;$'<+=:99>+?)@&'1%-)+9+ ! 119 Figure A 18: AmiE WT heatmap. AmiE WT 123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960Mutation MRHGDISSSNDTVGVAVVNYKMPRLHTAAEVLDNARKIAEMIVGMKQGLPGMDLVVFPEYSTOP *-2.87-0.22-1.15-2.05-0.93-1.66NaN-0.09-0.15-1.74-1.74-2.75-1.34-1.11 -1.68-1.94-1.92-1.96-1.82-0.58-0.78-1.93-2.84-1.79-0.94-1.22-1.69-2.65-1.87-0.27-1.70-1.89-1.47-1.80-1.25-0.75-0.53-1.82-2.00-0.76-2.29-2.16-1.79-1.99-1.97-0.55-0.64-1.41-1.46-1.34-2.02-1.62-0.56-1.61-1.60-2.37NaN-1.55-0.56-0.40F-1.86-0.85-0.74-1.28-1.54-0.27-1.00NaNNaN-1.03-2.42NaN-2.19-2.34-1.61-2.34-1.02-0.97-1.56-0.35-0.53-1.26-1.64-2.16-0.15-0.80-0.76-0.81-0.51-1.25-0.27-0.55-0.44-1.69-1.36-0.29-0.75-0.42NaN-1.08-0.14-0.47-0.52-0.81-0.61-1.19-0.50-1.39-0.13-1.28-2.08-0.60-1.92-1.71-0.26NaN0.00-1.83-1.48-0.53WNaNNaNNaN-2.05-0.50NaN-0.83-0.61NaN-1.38-1.96-1.01-1.25-1.92-2.57-1.15-1.22-1.74-1.43-1.13-3.52-1.59-2.16-1.42-1.27-0.57-0.92NaN-0.58-2.24-1.46-0.84-0.90NaNNaN-0.32-0.97NaN-0.83-1.14-0.81-2.50-0.26-0.92-0.90-1.65-1.57-1.36-0.26-0.76-1.32-2.14-2.42NaN-1.64NaNNaN-1.08-1.32NaNYNaNNaN-0.28-1.60-0.41-1.96-1.42-1.280.06-0.52-0.37-0.94-1.48-1.64-1.90-1.82-1.41-1.92-0.550.00-1.42-1.77-1.21-1.50-0.97-0.39-0.85-0.96-0.42-1.42-1.47-0.45-0.13-1.01-1.52-0.30-0.25-1.91NaN-0.87-0.27-1.01-0.22-0.84-0.98-1.780.00-0.96-0.08-0.42-1.86-1.25-0.34-1.40-2.13-2.65-0.39-2.37-1.810.00P-2.07-1.08-0.96-1.35-1.99-1.34-1.75-0.34-0.210.06-1.61-1.38-2.37-1.38-1.55-1.15-1.75-1.77-1.24-1.42-0.11 -1.070.00-1.36-1.28-0.69-0.530.15-0.19-1.68-1.93-0.35-1.20-1.82-1.30-0.95-1.86-1.75-0.44-1.53-1.40-1.65-0.61-0.49-1.69-1.51-0.64-1.53-0.730.00-1.92-1.51-1.45-0.53-1.19-1.32-2.800.00-1.09-1.48START M0.00-1.57NaN-1.25-0.98-0.32NaNNaN0.17-0.22-1.94-1.28-1.28NaN-2.27-0.85-0.10-0.13-1.40-1.25-0.370.00-1.04-0.10-0.38-0.48-0.85-0.35-0.18-0.69-1.14-0.02-0.24-1.31-1.64-0.180.04-0.16NaN-0.650.00-0.24-0.11 -0.530.00-0.45-0.20-1.580.05-1.10NaN0.00-1.64-0.30-1.10-0.13-1.05NaN-2.67NaNI-0.12-0.340.27-0.49-1.870.00-0.20-0.190.28-0.14-1.46-0.23-0.10-1.66-0.32-1.49-1.56-0.41-0.95-1.77-0.44-0.27-1.98-0.48-0.18-1.12-0.69-0.610.02-1.69-0.250.03-0.18-0.74-3.29-0.57-0.420.00NaN-1.29-0.280.000.03-1.61-0.34-2.10-0.37-1.71-0.13-0.90-1.33-0.15-1.54-0.54-0.12-0.14-0.37-0.81-1.35-1.25L-0.33-0.950.24-1.04-1.60-0.44-0.60-0.490.14-0.23-1.62-1.16-0.43-1.65-0.54-0.99-1.13-0.72-1.85-1.47-1.17-0.64-0.34-0.860.00-0.57-0.47-0.58-0.34-1.22-0.600.00-0.22-1.47-2.12-0.25-0.32-0.21-0.64-0.82-0.23-0.17-0.16-0.90-0.27-1.54-0.39-1.690.00-0.40-1.150.10-1.750.00-0.81-0.64-0.22-0.72-1.57-1.48V-0.29NaN0.32-0.33-0.53-0.18-2.16-0.74-0.36-0.26-0.46-1.340.00-0.230.00-0.040.000.00-1.95-1.57-0.38-0.52-0.53-0.98-0.28-1.31-0.72-0.05-0.15-0.390.00-0.10-0.04-1.96-0.15-0.13-0.29-0.30-0.01-0.42-0.140.050.00-0.85-0.42-1.37-0.14-0.86-0.48-0.93-0.39-0.35-0.31-0.610.000.00-0.44-1.72-0.53NaNA-1.70-1.64-0.60-0.10-0.33-1.56-1.220.100.320.23-0.99-0.16-0.59-0.65-0.670.00-0.43-0.39-1.70-1.40-0.19-1.33-0.87-0.66-0.77-0.86-0.270.000.00-0.12-0.57-0.28-0.08-1.050.00-0.14-0.06-1.470.00-0.14-0.77-0.78-0.18-0.22-1.11 -0.63-0.05-0.56-0.71-0.30-0.91-1.23-1.09-1.76-0.44-0.42-1.39-0.85-1.55NaNG-1.36-0.08-1.200.00-0.78-2.230.190.280.28-0.06-0.53-0.55-0.900.00-1.08-0.68-0.95-0.42-1.43-1.04-0.66-1.87-1.40-0.77-0.27-0.58-0.26-0.17-0.06-0.17-0.80-0.51-0.10-0.88-0.31-0.22-0.43-1.39-0.17-0.19-1.30-1.78-0.180.00-1.75-0.87-0.150.00-1.36-1.290.00-1.76-0.38-1.39-1.10-0.54-1.89-2.34-0.56NaNCNaNNaN-0.36-0.180.10-0.70-0.18NaN0.34-1.28-0.810.08-0.780.00-0.49-0.01-0.93-0.68-3.64-0.17-0.61-0.74-1.94-0.37-0.11 -1.10-0.07NaN-0.21-0.18-0.46-0.30-0.27-1.20-0.41-0.11 -0.19-1.77-0.90-0.49-0.51-0.81-0.15-0.27-1.10-1.450.07-0.64-0.56-0.94-0.31-0.43-1.90-0.97-0.31-0.44-0.54-1.94-1.77-0.26S-1.71-0.29-0.290.16-1.51-1.090.000.000.00-0.20-1.24-0.22-1.84-0.62-1.80-0.37-2.05-1.72-0.90-1.24-0.19-1.59-0.17-0.37-0.11 -0.650.000.02-0.02-0.65-1.21-0.43-0.26-0.27-0.59-0.11 -0.17-0.75-0.21-0.12-1.06-1.01-0.26-0.30-0.88-0.83-0.16-0.94-0.91-0.16-0.62-1.70-1.53-1.27-1.81-1.57-0.45-0.82-1.53-1.05T-0.43-0.620.28-0.99-1.71-0.54-0.490.200.250.17-0.980.00-1.67-1.30-1.02-0.41-1.19-1.29-1.06-1.92-0.05-0.66-0.49-0.63-0.54-0.560.000.01-0.12-1.12-0.720.03-0.08-1.25-0.51-0.12-0.22-0.67-0.29-0.17-0.35-0.32-0.22-0.89-0.65-0.80-0.19-1.77-0.73-0.36-0.94-0.47-1.10-1.53-1.03-1.33-1.48-0.62-2.18-0.83NNaNNaN-0.07-1.18-0.44-0.49-0.46NaN0.160.00-0.50-0.25-2.41-1.21-2.14-1.42-0.54-1.860.00-1.12-0.58-1.64-3.52-0.87-0.59-0.240.05-0.09-0.29-1.16-2.03-0.29-0.170.00-1.500.00-0.09-1.00-1.11 -0.03-1.52-0.41-0.13-0.24-1.83-0.42-0.26-0.62-1.11 -0.81-1.56-2.52-0.55-2.47-1.50NaN-1.57-1.81-1.69-0.69Q-1.67-1.420.31-1.10-0.94-1.67-1.34-1.040.340.17-1.16-0.67-2.35-1.19-1.96-1.37-1.58-1.89-0.38-1.86-0.47-2.05-0.34-0.72-0.33-0.18-0.510.07-0.12-0.06-1.92-0.050.00-0.69-2.37-0.20-0.05-1.00-0.54-0.24-0.16-2.070.01-0.48-1.32-0.530.00-2.43-0.84-0.53-1.36-1.74-1.70-0.72-1.18-1.57-1.43-2.87-0.97-0.86DNaN-0.67-0.59-0.200.00-1.57-1.80-0.900.26-0.140.00-1.70-2.08-0.16-1.84-0.43-2.11 -1.85-0.70-0.68-1.23-2.13-1.26-1.35-1.51-0.02-0.43-0.37-0.08-0.06-0.57-1.610.00-0.61-0.60-0.63-0.31-2.01-2.62-0.01-0.53-1.86-0.32-0.48-1.90-1.93-0.39-0.69-1.89-2.32-0.17-1.290.00-2.77-0.37NaN-1.42-2.05-0.50-0.27E-1.55NaN-1.10-1.01-0.46-1.57-1.20-0.90-0.41-0.37-0.42-0.36-0.49-1.42-0.58-1.72-0.49-0.49-1.84-2.42-0.18-1.59-1.46-1.64-1.41-0.61-0.59-0.43-0.060.00-1.69-0.260.06-0.27-1.00-0.24-0.13-2.02-0.140.00NaN-1.75-0.06-0.93-1.49-0.29-0.31-1.62-3.33-1.83-2.32-1.86-0.19-3.19-3.13-0.57NaNNaN0.00NaNH-5.77NaN0.00NaN-0.74-1.44-1.26-3.030.360.06-0.74-1.00-5.60NaN-1.11 -2.02-1.59-0.57-1.00-0.60-0.79-1.93-1.08-0.37-0.980.00-0.310.02-0.18-0.57-1.89-0.30-0.20-0.50-1.780.04-0.23-1.54-1.34-0.25-0.50-1.88-0.11 -0.23-0.90-1.33-0.16-1.12-0.74-0.97-1.70-1.64-1.18-0.51-1.50-2.11 -1.50-0.24-1.70-0.30K-0.44-0.47-1.01-2.37-1.69-1.69-2.180.30-0.630.15-1.66-1.15-2.30-1.92-1.57-1.38-1.67-1.43-0.46-1.410.00-0.87-0.31-0.57-1.58-0.52-0.240.28-0.16-0.58-1.57-0.02-0.29-0.53-1.81-0.050.00-1.28NaN-0.22-0.58-1.70-0.26-0.15-0.920.00-0.07-1.32-1.07-0.72-0.88-1.13-1.54NaNNaN-1.70NaNNaN-0.49-1.83R-0.690.00-0.56-0.86-1.39-0.92-0.23-0.39-1.380.11 -1.23-2.34-1.62-0.76-1.55-2.01-1.68-1.75-1.79-1.54-0.16-1.46-0.940.00-1.73-0.45-0.360.27-0.16-1.16-1.69-0.03-0.24-1.76-2.000.00-0.08-1.81-1.34-0.19-0.63-1.60-0.41-0.59-1.37-0.41-0.07-1.28-1.17-0.80-1.06-0.93-1.74-1.08-2.14-1.99-2.16-1.04-2.40-4.43AmiE WT 616263646566676869707172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110 111112 113 114 115 116 117 118 119 120Mutation SLQGIMYDPAEMMETAVAIPGEETEIFSRACRKANVWGVFSLTGERHEEHPRKAPYNTLVSTOP 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0.00-0.60-1.07-1.34-1.06-2.09-1.59-0.81-1.82-1.43-0.37-0.83-1.170.08-1.60-1.60-0.44-2.030.010.00-1.55-1.65-1.67-1.67-0.14-2.00-0.25-0.91-0.93-1.51-0.30-2.07-1.31NaN-1.75-1.77-1.45-1.76-1.45-2.65-1.33-2.26-0.17-1.09-2.95-2.37-4.09-2.74-3.69NaN-1.99DNaN-1.890.00-0.20-0.11 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WT 181182183184185186187188189190191192193194195196197198199200201202203204205206207208209210211 212213214215216217218219220221222223224225226227228229230231232233234235236237238239240Mutation KGAELIVRCQGYMYPAKDQQVMMAKAMAWANNCYVAVANAAGFDGVYSYFGHSAIIGFDGSTOP *-0.53-2.00-3.13-0.25-0.95-2.92NaN-2.51-0.69-0.50NaN-0.50-2.32-0.16NaN-2.33-1.01-1.37-0.58-0.53NaN-2.47-2.58-1.79-0.71-1.25NaN-2.90-0.08-2.98-3.36-3.84-0.43-0.39-2.59NaN-2.08-1.73-4.45-1.78-1.95-1.99-2.53-2.22-2.89-3.05-0.39-1.25-0.71-2.94####-2.52-2.45-1.64-3.77-2.99-2.86-2.67NaN-3.58F-0.93-2.33-5.53-2.74-0.12-0.50-0.63-2.70-0.46-3.62-1.68-0.05-2.33-0.61NaN-3.19-8.26-1.16-2.56-3.27-0.50-1.47-1.33-2.45NaN-3.16-1.79-1.57-0.58-2.07-3.84-1.00-0.28-0.66-0.65-1.97-2.11 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M0.00-2.15-1.78-3.88-0.49-0.45-2.86NaN-3.480.20NaNNaN0.00NaNNaN-3.29-1.32-1.66-0.54-3.530.370.000.00-2.21-0.17NaN0.00-2.09NaN-1.75NaNNaNNaNNaNNaNNaN-1.38-1.96-0.97-1.89NaNNaN-1.59NaNNaN-0.54-3.62NaN-2.59NaN-0.72NaN-3.970.33-0.57-0.72NaN-1.23-1.75NaNINaNNaN-2.09NaN-1.310.00-0.13-7.44-2.39-2.40-2.33-2.10-0.24-1.79NaN-2.13-0.67-1.41-0.53-0.860.20-0.06-0.44-1.81-2.93-1.41-0.28-2.11 NaNNaN-0.14-0.49-0.99-2.30-0.63NaN-0.40-3.02-0.87-0.06-2.15-2.52-1.45-2.98-1.80-0.15-2.74-0.20-2.61-0.54NaN-3.10-0.89-0.460.000.00NaN-0.79-4.00-1.66L-2.36NaN-2.00-2.220.00-0.31-1.54-0.50-2.39-0.54-2.13-2.25-0.39-2.26-0.43-3.31-1.20-0.69-0.62-1.11 0.16-0.03-0.62-2.63-1.31-1.90-0.31-2.08-0.35-2.35-3.09-0.83-1.17-2.67-1.81-2.13-0.65-1.87-3.15-0.72-3.54-2.66-0.73NaN-2.10-0.94-1.63-6.36-3.32-0.25-3.96-0.48-2.48-0.18-0.66-0.42-2.66-0.53-4.16-3.96V-2.16-0.52-0.10-0.45-0.35-0.160.00NaN-1.33-3.11 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-0.64-0.83-0.42-0.67-0.32-1.57-2.33-0.24-2.21-2.190.00-2.75-5.15-0.67-2.02-0.71-0.70G-1.780.00-1.27-0.56-2.51-2.35-0.50-0.58-1.05-1.310.00-3.59-0.85-1.92NaN-0.44NaN-0.23NaN-1.99-0.18-0.72-2.25-0.41-2.32-0.87-1.75-0.93-0.90-0.61NaN-0.27-0.56-2.68-0.95-0.49-1.07-0.61-1.96-0.37-0.480.00-3.73-0.320.00-0.36NaN0.05-3.54-2.210.00-0.91-0.53-0.76-3.31-2.740.00-2.99-0.120.00C-2.95-0.42-1.36-4.26-1.35-1.91-0.13-0.170.00-0.30-0.53-0.54-1.86-0.12-1.43-0.30-1.20-1.22-1.25-1.090.13-0.42-2.470.23-2.210.15-2.09-1.44-0.01-0.46-1.75-0.370.00-0.69-1.64NaN-0.43-1.40-3.25-0.71-0.30-0.31-0.48-4.43-0.15-0.12-0.17-0.20-0.20-0.18-0.14-0.68-0.610.28-3.38-2.89-0.53-0.60-1.33-0.32S-1.98-1.26-0.39-5.84-0.58-1.11 NaN-0.740.04-0.04-0.42-0.95-1.54-0.37-0.97-0.25-1.58-1.23-1.03-0.140.02-0.48-2.790.10-1.39-0.21-3.84-0.66-0.90-0.68-0.61-0.33-0.62-1.91-2.66-0.32-1.82-0.54-0.37-0.55-0.02-1.30-0.85-1.71-0.47-1.03-0.590.00-1.14-0.23-0.26-1.990.00-0.04-1.48-0.710.08-0.88-3.47-0.48T-1.18-2.30-0.48-2.64-2.99-0.80-2.96-2.35-0.77-0.49-3.20-2.36-0.38-2.36-0.68-0.26-0.89-0.32-0.39-1.840.20-0.11 -0.81-0.17-0.860.02-0.64-0.64NaN-0.44-1.48-0.25-0.51-2.29-2.61-0.18-0.46-0.12-0.91-0.510.04-3.09-0.81-2.68-3.02-0.34-3.06-0.37-2.90-0.78-1.78-1.60-1.620.07-0.85-0.78-2.68-3.10-2.15-2.80N-0.38-2.70-1.08-2.21NaN-0.23-3.48-2.54-1.35-0.02NaN-1.09-1.77-0.10-1.11 -0.84-0.43-0.46-1.33-1.21-0.11 -0.12-3.78-0.28-0.57-1.25-2.86-2.23-2.09-2.390.000.00-1.60-1.04-2.71-3.22NaN-4.120.00-2.34NaNNaN-0.97-0.48-2.80-0.59-0.57-0.69-0.63-2.36-2.02-0.13-0.71-1.55-0.74-0.66-1.11 -3.22-0.83-2.23Q-0.66-2.02-3.44-1.26-2.26NaN-2.05-1.53-1.800.00NaN-3.38-2.03-0.15-2.99-1.02-0.24-0.600.000.000.01-0.35-2.26-1.50-0.62-1.80-1.58-3.13NaN-3.08-2.440.00-2.04-2.10NaN-3.01-0.08-5.05-2.97-3.92-2.74-2.83-0.20-2.19NaN-1.24-1.29-1.73-3.19NaN-3.86-0.10-3.440.06-3.19-1.96-4.92-1.95-2.55-2.78DNaN-0.54-2.15-0.37-1.88-0.81-0.19-1.67-1.23-3.29-0.15-0.44NaN-0.19NaN-0.22-2.610.00-1.11 -2.52-0.51-1.95NaN-0.26NaN-1.50-2.43-0.73-1.49-2.15-0.54-0.25-2.49-0.68-0.49-0.25-3.66NaN-0.38-0.28-2.35-0.42-2.380.00-0.52-0.81-0.35-2.76-0.13-2.02-0.54-0.45-2.26-0.81NaNNaN-0.43NaN0.00-0.32E-0.27-2.95-0.560.00-2.82-3.29-2.09-1.89NaN-0.35NaN-2.68-2.12-3.62NaNNaN-0.28-0.01-0.73-0.80-0.74-1.67NaN-2.15-0.33-0.88-3.29-2.06-2.15-0.23-1.82-1.03-2.77-2.98-2.96-2.09-0.82-0.22-3.55-1.73-0.27-4.04-2.05-0.22-2.22NaNNaN-1.92-1.82NaNNaN-2.02-3.31-2.06-1.98-2.12-3.62-1.41-0.57-1.96H-1.38-3.27-2.48-2.27-4.25-1.64-5.01-0.41NaN-0.19-1.75-0.21-5.73-0.53-0.20-3.16-1.63-0.61-0.10-0.32-0.64-0.08-2.12-1.61-1.13-2.11 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-2.120.00-3.91-0.52-0.96-0.38-0.40-1.45-0.32-0.18-0.86-0.97-0.47-1.530.00-1.290.050.000.00-0.220.13-0.020.09-0.35-0.19-0.09-0.260.16-0.16-0.210.23-0.20-0.24N-2.86-2.72-1.44-2.87-0.54-0.90-0.22NaN-2.02-0.01-0.15-3.59-0.33-0.45NaN-2.13-0.430.00-3.18-2.09-0.13-1.88-0.55-1.02-0.05-0.15-0.97-0.16-0.32-0.03-0.05-0.10-0.30-0.58-0.11 0.28-0.04-0.210.30-0.15-0.06Q-0.29NaN-0.71-2.88-2.60-0.74-0.21NaN-2.60-0.04-1.41-2.01-0.47-0.18-2.06-0.95-0.08-0.02-1.97-0.40-0.06-0.46NaN-1.590.04-0.17-1.65-0.11 -0.880.130.00-1.01-0.08-0.46-0.100.24-0.14-0.290.11 -0.19-0.14D-1.44-3.44-0.04-3.44-0.61-1.60-1.24NaN-0.51-1.160.00-0.81-0.10-1.62-1.92-2.33-0.10-0.03-3.62-0.25-2.15-2.35-2.30NaN-0.33-0.29-1.38-0.13-0.340.140.11 -0.19-0.58-0.050.090.330.17-0.090.310.170.13E-2.65NaN0.00NaN-1.69-3.17-1.35NaN-1.90-0.42-0.25-1.200.00-0.27-0.25-2.750.00-0.41-0.720.00-0.52-1.48-3.17NaN-0.51-0.13-1.55-0.09-0.950.160.21-0.24-0.25-0.190.150.310.27-0.620.200.000.03H-2.77-2.23-2.27-1.88-0.15-0.16-0.06-3.22NaN-0.41-0.63-0.24-1.11 -1.31-2.51-2.22-0.63-0.43-3.42-1.37-0.25-2.06-4.95-0.15-0.11 0.22-1.92-0.23-0.77-0.16-0.15-0.43-0.49-0.20-0.380.07-0.07-0.250.18-0.24-0.20KNaN-2.24-0.71-2.35-1.140.09-1.21-1.14-1.19-0.13-1.42-1.69-0.090.00-1.89-1.00-0.21-0.40-1.97-0.470.15-2.60NaN-0.430.16-0.09-0.15-0.35-0.930.06-0.06-0.17-0.11 -0.47-0.120.21-0.18-0.390.03-0.120.04R-1.29-2.50-2.44-2.91NaN0.00-1.40-0.11 -2.21-0.31-2.48-1.84-0.89-0.13-2.490.00-0.34-0.54-2.27-0.830.00-0.79-2.770.00-0.06-0.24-0.75-0.22-1.04-0.02-0.34-0.41-0.26-0.97-0.310.00-0.18-0.37-0.04-0.15-0.13Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged AmiE WT 123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960Mutation MRHGDISSSNDTVGVAVVNYKMPRLHTAAEVLDNARKIAEMIVGMKQGLPGMDLVVFPEYSTOP *-2.87-0.22-1.15-2.05-0.93-1.66NaN-0.09-0.15-1.74-1.74-2.75-1.34-1.11 -1.68-1.94-1.92-1.96-1.82-0.58-0.78-1.93-2.84-1.79-0.94-1.22-1.69-2.65-1.87-0.27-1.70-1.89-1.47-1.80-1.25-0.75-0.53-1.82-2.00-0.76-2.29-2.16-1.79-1.99-1.97-0.55-0.64-1.41-1.46-1.34-2.02-1.62-0.56-1.61-1.60-2.37NaN-1.55-0.56-0.40F-1.86-0.85-0.74-1.28-1.54-0.27-1.00NaNNaN-1.03-2.42NaN-2.19-2.34-1.61-2.34-1.02-0.97-1.56-0.35-0.53-1.26-1.64-2.16-0.15-0.80-0.76-0.81-0.51-1.25-0.27-0.55-0.44-1.69-1.36-0.29-0.75-0.42NaN-1.08-0.14-0.47-0.52-0.81-0.61-1.19-0.50-1.39-0.13-1.28-2.08-0.60-1.92-1.71-0.26NaN0.00-1.83-1.48-0.53WNaNNaNNaN-2.05-0.50NaN-0.83-0.61NaN-1.38-1.96-1.01-1.25-1.92-2.57-1.15-1.22-1.74-1.43-1.13-3.52-1.59-2.16-1.42-1.27-0.57-0.92NaN-0.58-2.24-1.46-0.84-0.90NaNNaN-0.32-0.97NaN-0.83-1.14-0.81-2.50-0.26-0.92-0.90-1.65-1.57-1.36-0.26-0.76-1.32-2.14-2.42NaN-1.64NaNNaN-1.08-1.32NaNYNaNNaN-0.28-1.60-0.41-1.96-1.42-1.280.06-0.52-0.37-0.94-1.48-1.64-1.90-1.82-1.41-1.92-0.550.00-1.42-1.77-1.21-1.50-0.97-0.39-0.85-0.96-0.42-1.42-1.47-0.45-0.13-1.01-1.52-0.30-0.25-1.91NaN-0.87-0.27-1.01-0.22-0.84-0.98-1.780.00-0.96-0.08-0.42-1.86-1.25-0.34-1.40-2.13-2.65-0.39-2.37-1.810.00P-2.07-1.08-0.96-1.35-1.99-1.34-1.75-0.34-0.210.06-1.61-1.38-2.37-1.38-1.55-1.15-1.75-1.77-1.24-1.42-0.11 -1.070.00-1.36-1.28-0.69-0.530.15-0.19-1.68-1.93-0.35-1.20-1.82-1.30-0.95-1.86-1.75-0.44-1.53-1.40-1.65-0.61-0.49-1.69-1.51-0.64-1.53-0.730.00-1.92-1.51-1.45-0.53-1.19-1.32-2.800.00-1.09-1.48START M0.00-1.57NaN-1.25-0.98-0.32NaNNaN0.17-0.22-1.94-1.28-1.28NaN-2.27-0.85-0.10-0.13-1.40-1.25-0.370.00-1.04-0.10-0.38-0.48-0.85-0.35-0.18-0.69-1.14-0.02-0.24-1.31-1.64-0.180.04-0.16NaN-0.650.00-0.24-0.11 -0.530.00-0.45-0.20-1.580.05-1.10NaN0.00-1.64-0.30-1.10-0.13-1.05NaN-2.67NaNI-0.12-0.340.27-0.49-1.870.00-0.20-0.190.28-0.14-1.46-0.23-0.10-1.66-0.32-1.49-1.56-0.41-0.95-1.77-0.44-0.27-1.98-0.48-0.18-1.12-0.69-0.610.02-1.69-0.250.03-0.18-0.74-3.29-0.57-0.420.00NaN-1.29-0.280.000.03-1.61-0.34-2.10-0.37-1.71-0.13-0.90-1.33-0.15-1.54-0.54-0.12-0.14-0.37-0.81-1.35-1.25L-0.33-0.950.24-1.04-1.60-0.44-0.60-0.490.14-0.23-1.62-1.16-0.43-1.65-0.54-0.99-1.13-0.72-1.85-1.47-1.17-0.64-0.34-0.860.00-0.57-0.47-0.58-0.34-1.22-0.600.00-0.22-1.47-2.12-0.25-0.32-0.21-0.64-0.82-0.23-0.17-0.16-0.90-0.27-1.54-0.39-1.690.00-0.40-1.150.10-1.750.00-0.81-0.64-0.22-0.72-1.57-1.48V-0.29NaN0.32-0.33-0.53-0.18-2.16-0.74-0.36-0.26-0.46-1.340.00-0.230.00-0.040.000.00-1.95-1.57-0.38-0.52-0.53-0.98-0.28-1.31-0.72-0.05-0.15-0.390.00-0.10-0.04-1.96-0.15-0.13-0.29-0.30-0.01-0.42-0.140.050.00-0.85-0.42-1.37-0.14-0.86-0.48-0.93-0.39-0.35-0.31-0.610.000.00-0.44-1.72-0.53NaNA-1.70-1.64-0.60-0.10-0.33-1.56-1.220.100.320.23-0.99-0.16-0.59-0.65-0.670.00-0.43-0.39-1.70-1.40-0.19-1.33-0.87-0.66-0.77-0.86-0.270.000.00-0.12-0.57-0.28-0.08-1.050.00-0.14-0.06-1.470.00-0.14-0.77-0.78-0.18-0.22-1.11 -0.63-0.05-0.56-0.71-0.30-0.91-1.23-1.09-1.76-0.44-0.42-1.39-0.85-1.55NaNG-1.36-0.08-1.200.00-0.78-2.230.190.280.28-0.06-0.53-0.55-0.900.00-1.08-0.68-0.95-0.42-1.43-1.04-0.66-1.87-1.40-0.77-0.27-0.58-0.26-0.17-0.06-0.17-0.80-0.51-0.10-0.88-0.31-0.22-0.43-1.39-0.17-0.19-1.30-1.78-0.180.00-1.75-0.87-0.150.00-1.36-1.290.00-1.76-0.38-1.39-1.10-0.54-1.89-2.34-0.56NaNCNaNNaN-0.36-0.180.10-0.70-0.18NaN0.34-1.28-0.810.08-0.780.00-0.49-0.01-0.93-0.68-3.64-0.17-0.61-0.74-1.94-0.37-0.11 -1.10-0.07NaN-0.21-0.18-0.46-0.30-0.27-1.20-0.41-0.11 -0.19-1.77-0.90-0.49-0.51-0.81-0.15-0.27-1.10-1.450.07-0.64-0.56-0.94-0.31-0.43-1.90-0.97-0.31-0.44-0.54-1.94-1.77-0.26S-1.71-0.29-0.290.16-1.51-1.090.000.000.00-0.20-1.24-0.22-1.84-0.62-1.80-0.37-2.05-1.72-0.90-1.24-0.19-1.59-0.17-0.37-0.11 -0.650.000.02-0.02-0.65-1.21-0.43-0.26-0.27-0.59-0.11 -0.17-0.75-0.21-0.12-1.06-1.01-0.26-0.30-0.88-0.83-0.16-0.94-0.91-0.16-0.62-1.70-1.53-1.27-1.81-1.57-0.45-0.82-1.53-1.05T-0.43-0.620.28-0.99-1.71-0.54-0.490.200.250.17-0.980.00-1.67-1.30-1.02-0.41-1.19-1.29-1.06-1.92-0.05-0.66-0.49-0.63-0.54-0.560.000.01-0.12-1.12-0.720.03-0.08-1.25-0.51-0.12-0.22-0.67-0.29-0.17-0.35-0.32-0.22-0.89-0.65-0.80-0.19-1.77-0.73-0.36-0.94-0.47-1.10-1.53-1.03-1.33-1.48-0.62-2.18-0.83NNaNNaN-0.07-1.18-0.44-0.49-0.46NaN0.160.00-0.50-0.25-2.41-1.21-2.14-1.42-0.54-1.860.00-1.12-0.58-1.64-3.52-0.87-0.59-0.240.05-0.09-0.29-1.16-2.03-0.29-0.170.00-1.500.00-0.09-1.00-1.11 -0.03-1.52-0.41-0.13-0.24-1.83-0.42-0.26-0.62-1.11 -0.81-1.56-2.52-0.55-2.47-1.50NaN-1.57-1.81-1.69-0.69Q-1.67-1.420.31-1.10-0.94-1.67-1.34-1.040.340.17-1.16-0.67-2.35-1.19-1.96-1.37-1.58-1.89-0.38-1.86-0.47-2.05-0.34-0.72-0.33-0.18-0.510.07-0.12-0.06-1.92-0.050.00-0.69-2.37-0.20-0.05-1.00-0.54-0.24-0.16-2.070.01-0.48-1.32-0.530.00-2.43-0.84-0.53-1.36-1.74-1.70-0.72-1.18-1.57-1.43-2.87-0.97-0.86DNaN-0.67-0.59-0.200.00-1.57-1.80-0.900.26-0.140.00-1.70-2.08-0.16-1.84-0.43-2.11 -1.85-0.70-0.68-1.23-2.13-1.26-1.35-1.51-0.02-0.43-0.37-0.08-0.06-0.57-1.610.00-0.61-0.60-0.63-0.31-2.01-2.62-0.01-0.53-1.86-0.32-0.48-1.90-1.93-0.39-0.69-1.89-2.32-0.17-1.290.00-2.77-0.37NaN-1.42-2.05-0.50-0.27E-1.55NaN-1.10-1.01-0.46-1.57-1.20-0.90-0.41-0.37-0.42-0.36-0.49-1.42-0.58-1.72-0.49-0.49-1.84-2.42-0.18-1.59-1.46-1.64-1.41-0.61-0.59-0.43-0.060.00-1.69-0.260.06-0.27-1.00-0.24-0.13-2.02-0.140.00NaN-1.75-0.06-0.93-1.49-0.29-0.31-1.62-3.33-1.83-2.32-1.86-0.19-3.19-3.13-0.57NaNNaN0.00NaNH-5.77NaN0.00NaN-0.74-1.44-1.26-3.030.360.06-0.74-1.00-5.60NaN-1.11 -2.02-1.59-0.57-1.00-0.60-0.79-1.93-1.08-0.37-0.980.00-0.310.02-0.18-0.57-1.89-0.30-0.20-0.50-1.780.04-0.23-1.54-1.34-0.25-0.50-1.88-0.11 -0.23-0.90-1.33-0.16-1.12-0.74-0.97-1.70-1.64-1.18-0.51-1.50-2.11 -1.50-0.24-1.70-0.30K-0.44-0.47-1.01-2.37-1.69-1.69-2.180.30-0.630.15-1.66-1.15-2.30-1.92-1.57-1.38-1.67-1.43-0.46-1.410.00-0.87-0.31-0.57-1.58-0.52-0.240.28-0.16-0.58-1.57-0.02-0.29-0.53-1.81-0.050.00-1.28NaN-0.22-0.58-1.70-0.26-0.15-0.920.00-0.07-1.32-1.07-0.72-0.88-1.13-1.54NaNNaN-1.70NaNNaN-0.49-1.83R-0.690.00-0.56-0.86-1.39-0.92-0.23-0.39-1.380.11 -1.23-2.34-1.62-0.76-1.55-2.01-1.68-1.75-1.79-1.54-0.16-1.46-0.940.00-1.73-0.45-0.360.27-0.16-1.16-1.69-0.03-0.24-1.76-2.000.00-0.08-1.81-1.34-0.19-0.63-1.60-0.41-0.59-1.37-0.41-0.07-1.28-1.17-0.80-1.06-0.93-1.74-1.08-2.14-1.99-2.16-1.04-2.40-4.43AmiE WT 616263646566676869707172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110 111112 113 114 115 116 117 118 119 120Mutation SLQGIMYDPAEMMETAVAIPGEETEIFSRACRKANVWGVFSLTGERHEEHPRKAPYNTLVSTOP 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0.00-0.60-1.07-1.34-1.06-2.09-1.59-0.81-1.82-1.43-0.37-0.83-1.170.08-1.60-1.60-0.44-2.030.010.00-1.55-1.65-1.67-1.67-0.14-2.00-0.25-0.91-0.93-1.51-0.30-2.07-1.31NaN-1.75-1.77-1.45-1.76-1.45-2.65-1.33-2.26-0.17-1.09-2.95-2.37-4.09-2.74-3.69NaN-1.99DNaN-1.890.00-0.20-0.11 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WT 181182183184185186187188189190191192193194195196197198199200201202203204205206207208209210211 212213214215216217218219220221222223224225226227228229230231232233234235236237238239240Mutation KGAELIVRCQGYMYPAKDQQVMMAKAMAWANNCYVAVANAAGFDGVYSYFGHSAIIGFDGSTOP *-0.53-2.00-3.13-0.25-0.95-2.92NaN-2.51-0.69-0.50NaN-0.50-2.32-0.16NaN-2.33-1.01-1.37-0.58-0.53NaN-2.47-2.58-1.79-0.71-1.25NaN-2.90-0.08-2.98-3.36-3.84-0.43-0.39-2.59NaN-2.08-1.73-4.45-1.78-1.95-1.99-2.53-2.22-2.89-3.05-0.39-1.25-0.71-2.94####-2.52-2.45-1.64-3.77-2.99-2.86-2.67NaN-3.58F-0.93-2.33-5.53-2.74-0.12-0.50-0.63-2.70-0.46-3.62-1.68-0.05-2.33-0.61NaN-3.19-8.26-1.16-2.56-3.27-0.50-1.47-1.33-2.45NaN-3.16-1.79-1.57-0.58-2.07-3.84-1.00-0.28-0.66-0.65-1.97-2.11 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M0.00-2.15-1.78-3.88-0.49-0.45-2.86NaN-3.480.20NaNNaN0.00NaNNaN-3.29-1.32-1.66-0.54-3.530.370.000.00-2.21-0.17NaN0.00-2.09NaN-1.75NaNNaNNaNNaNNaNNaN-1.38-1.96-0.97-1.89NaNNaN-1.59NaNNaN-0.54-3.62NaN-2.59NaN-0.72NaN-3.970.33-0.57-0.72NaN-1.23-1.75NaNINaNNaN-2.09NaN-1.310.00-0.13-7.44-2.39-2.40-2.33-2.10-0.24-1.79NaN-2.13-0.67-1.41-0.53-0.860.20-0.06-0.44-1.81-2.93-1.41-0.28-2.11 NaNNaN-0.14-0.49-0.99-2.30-0.63NaN-0.40-3.02-0.87-0.06-2.15-2.52-1.45-2.98-1.80-0.15-2.74-0.20-2.61-0.54NaN-3.10-0.89-0.460.000.00NaN-0.79-4.00-1.66L-2.36NaN-2.00-2.220.00-0.31-1.54-0.50-2.39-0.54-2.13-2.25-0.39-2.26-0.43-3.31-1.20-0.69-0.62-1.11 0.16-0.03-0.62-2.63-1.31-1.90-0.31-2.08-0.35-2.35-3.09-0.83-1.17-2.67-1.81-2.13-0.65-1.87-3.15-0.72-3.54-2.66-0.73NaN-2.10-0.94-1.63-6.36-3.32-0.25-3.96-0.48-2.48-0.18-0.66-0.42-2.66-0.53-4.16-3.96V-2.16-0.52-0.10-0.45-0.35-0.160.00NaN-1.33-3.11 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-0.64-0.83-0.42-0.67-0.32-1.57-2.33-0.24-2.21-2.190.00-2.75-5.15-0.67-2.02-0.71-0.70G-1.780.00-1.27-0.56-2.51-2.35-0.50-0.58-1.05-1.310.00-3.59-0.85-1.92NaN-0.44NaN-0.23NaN-1.99-0.18-0.72-2.25-0.41-2.32-0.87-1.75-0.93-0.90-0.61NaN-0.27-0.56-2.68-0.95-0.49-1.07-0.61-1.96-0.37-0.480.00-3.73-0.320.00-0.36NaN0.05-3.54-2.210.00-0.91-0.53-0.76-3.31-2.740.00-2.99-0.120.00C-2.95-0.42-1.36-4.26-1.35-1.91-0.13-0.170.00-0.30-0.53-0.54-1.86-0.12-1.43-0.30-1.20-1.22-1.25-1.090.13-0.42-2.470.23-2.210.15-2.09-1.44-0.01-0.46-1.75-0.370.00-0.69-1.64NaN-0.43-1.40-3.25-0.71-0.30-0.31-0.48-4.43-0.15-0.12-0.17-0.20-0.20-0.18-0.14-0.68-0.610.28-3.38-2.89-0.53-0.60-1.33-0.32S-1.98-1.26-0.39-5.84-0.58-1.11 NaN-0.740.04-0.04-0.42-0.95-1.54-0.37-0.97-0.25-1.58-1.23-1.03-0.140.02-0.48-2.790.10-1.39-0.21-3.84-0.66-0.90-0.68-0.61-0.33-0.62-1.91-2.66-0.32-1.82-0.54-0.37-0.55-0.02-1.30-0.85-1.71-0.47-1.03-0.590.00-1.14-0.23-0.26-1.990.00-0.04-1.48-0.710.08-0.88-3.47-0.48T-1.18-2.30-0.48-2.64-2.99-0.80-2.96-2.35-0.77-0.49-3.20-2.36-0.38-2.36-0.68-0.26-0.89-0.32-0.39-1.840.20-0.11 -0.81-0.17-0.860.02-0.64-0.64NaN-0.44-1.48-0.25-0.51-2.29-2.61-0.18-0.46-0.12-0.91-0.510.04-3.09-0.81-2.68-3.02-0.34-3.06-0.37-2.90-0.78-1.78-1.60-1.620.07-0.85-0.78-2.68-3.10-2.15-2.80N-0.38-2.70-1.08-2.21NaN-0.23-3.48-2.54-1.35-0.02NaN-1.09-1.77-0.10-1.11 -0.84-0.43-0.46-1.33-1.21-0.11 -0.12-3.78-0.28-0.57-1.25-2.86-2.23-2.09-2.390.000.00-1.60-1.04-2.71-3.22NaN-4.120.00-2.34NaNNaN-0.97-0.48-2.80-0.59-0.57-0.69-0.63-2.36-2.02-0.13-0.71-1.55-0.74-0.66-1.11 -3.22-0.83-2.23Q-0.66-2.02-3.44-1.26-2.26NaN-2.05-1.53-1.800.00NaN-3.38-2.03-0.15-2.99-1.02-0.24-0.600.000.000.01-0.35-2.26-1.50-0.62-1.80-1.58-3.13NaN-3.08-2.440.00-2.04-2.10NaN-3.01-0.08-5.05-2.97-3.92-2.74-2.83-0.20-2.19NaN-1.24-1.29-1.73-3.19NaN-3.86-0.10-3.440.06-3.19-1.96-4.92-1.95-2.55-2.78DNaN-0.54-2.15-0.37-1.88-0.81-0.19-1.67-1.23-3.29-0.15-0.44NaN-0.19NaN-0.22-2.610.00-1.11 -2.52-0.51-1.95NaN-0.26NaN-1.50-2.43-0.73-1.49-2.15-0.54-0.25-2.49-0.68-0.49-0.25-3.66NaN-0.38-0.28-2.35-0.42-2.380.00-0.52-0.81-0.35-2.76-0.13-2.02-0.54-0.45-2.26-0.81NaNNaN-0.43NaN0.00-0.32E-0.27-2.95-0.560.00-2.82-3.29-2.09-1.89NaN-0.35NaN-2.68-2.12-3.62NaNNaN-0.28-0.01-0.73-0.80-0.74-1.67NaN-2.15-0.33-0.88-3.29-2.06-2.15-0.23-1.82-1.03-2.77-2.98-2.96-2.09-0.82-0.22-3.55-1.73-0.27-4.04-2.05-0.22-2.22NaNNaN-1.92-1.82NaNNaN-2.02-3.31-2.06-1.98-2.12-3.62-1.41-0.57-1.96H-1.38-3.27-2.48-2.27-4.25-1.64-5.01-0.41NaN-0.19-1.75-0.21-5.73-0.53-0.20-3.16-1.63-0.61-0.10-0.32-0.64-0.08-2.12-1.61-1.13-2.11 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-2.120.00-3.91-0.52-0.96-0.38-0.40-1.45-0.32-0.18-0.86-0.97-0.47-1.530.00-1.290.050.000.00-0.220.13-0.020.09-0.35-0.19-0.09-0.260.16-0.16-0.210.23-0.20-0.24N-2.86-2.72-1.44-2.87-0.54-0.90-0.22NaN-2.02-0.01-0.15-3.59-0.33-0.45NaN-2.13-0.430.00-3.18-2.09-0.13-1.88-0.55-1.02-0.05-0.15-0.97-0.16-0.32-0.03-0.05-0.10-0.30-0.58-0.11 0.28-0.04-0.210.30-0.15-0.06Q-0.29NaN-0.71-2.88-2.60-0.74-0.21NaN-2.60-0.04-1.41-2.01-0.47-0.18-2.06-0.95-0.08-0.02-1.97-0.40-0.06-0.46NaN-1.590.04-0.17-1.65-0.11 -0.880.130.00-1.01-0.08-0.46-0.100.24-0.14-0.290.11 -0.19-0.14D-1.44-3.44-0.04-3.44-0.61-1.60-1.24NaN-0.51-1.160.00-0.81-0.10-1.62-1.92-2.33-0.10-0.03-3.62-0.25-2.15-2.35-2.30NaN-0.33-0.29-1.38-0.13-0.340.140.11 -0.19-0.58-0.050.090.330.17-0.090.310.170.13E-2.65NaN0.00NaN-1.69-3.17-1.35NaN-1.90-0.42-0.25-1.200.00-0.27-0.25-2.750.00-0.41-0.720.00-0.52-1.48-3.17NaN-0.51-0.13-1.55-0.09-0.950.160.21-0.24-0.25-0.190.150.310.27-0.620.200.000.03H-2.77-2.23-2.27-1.88-0.15-0.16-0.06-3.22NaN-0.41-0.63-0.24-1.11 -1.31-2.51-2.22-0.63-0.43-3.42-1.37-0.25-2.06-4.95-0.15-0.11 0.22-1.92-0.23-0.77-0.16-0.15-0.43-0.49-0.20-0.380.07-0.07-0.250.18-0.24-0.20KNaN-2.24-0.71-2.35-1.140.09-1.21-1.14-1.19-0.13-1.42-1.69-0.090.00-1.89-1.00-0.21-0.40-1.97-0.470.15-2.60NaN-0.430.16-0.09-0.15-0.35-0.930.06-0.06-0.17-0.11 -0.47-0.120.21-0.18-0.390.03-0.120.04R-1.29-2.50-2.44-2.91NaN0.00-1.40-0.11 -2.21-0.31-2.48-1.84-0.89-0.13-2.490.00-0.34-0.54-2.27-0.830.00-0.79-2.770.00-0.06-0.24-0.75-0.22-1.04-0.02-0.34-0.41-0.26-0.97-0.310.00-0.18-0.37-0.04-0.15-0.13Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged AmiE WT 123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960Mutation MRHGDISSSNDTVGVAVVNYKMPRLHTAAEVLDNARKIAEMIVGMKQGLPGMDLVVFPEYSTOP *-2.87-0.22-1.15-2.05-0.93-1.66NaN-0.09-0.15-1.74-1.74-2.75-1.34-1.11 -1.68-1.94-1.92-1.96-1.82-0.58-0.78-1.93-2.84-1.79-0.94-1.22-1.69-2.65-1.87-0.27-1.70-1.89-1.47-1.80-1.25-0.75-0.53-1.82-2.00-0.76-2.29-2.16-1.79-1.99-1.97-0.55-0.64-1.41-1.46-1.34-2.02-1.62-0.56-1.61-1.60-2.37NaN-1.55-0.56-0.40F-1.86-0.85-0.74-1.28-1.54-0.27-1.00NaNNaN-1.03-2.42NaN-2.19-2.34-1.61-2.34-1.02-0.97-1.56-0.35-0.53-1.26-1.64-2.16-0.15-0.80-0.76-0.81-0.51-1.25-0.27-0.55-0.44-1.69-1.36-0.29-0.75-0.42NaN-1.08-0.14-0.47-0.52-0.81-0.61-1.19-0.50-1.39-0.13-1.28-2.08-0.60-1.92-1.71-0.26NaN0.00-1.83-1.48-0.53WNaNNaNNaN-2.05-0.50NaN-0.83-0.61NaN-1.38-1.96-1.01-1.25-1.92-2.57-1.15-1.22-1.74-1.43-1.13-3.52-1.59-2.16-1.42-1.27-0.57-0.92NaN-0.58-2.24-1.46-0.84-0.90NaNNaN-0.32-0.97NaN-0.83-1.14-0.81-2.50-0.26-0.92-0.90-1.65-1.57-1.36-0.26-0.76-1.32-2.14-2.42NaN-1.64NaNNaN-1.08-1.32NaNYNaNNaN-0.28-1.60-0.41-1.96-1.42-1.280.06-0.52-0.37-0.94-1.48-1.64-1.90-1.82-1.41-1.92-0.550.00-1.42-1.77-1.21-1.50-0.97-0.39-0.85-0.96-0.42-1.42-1.47-0.45-0.13-1.01-1.52-0.30-0.25-1.91NaN-0.87-0.27-1.01-0.22-0.84-0.98-1.780.00-0.96-0.08-0.42-1.86-1.25-0.34-1.40-2.13-2.65-0.39-2.37-1.810.00P-2.07-1.08-0.96-1.35-1.99-1.34-1.75-0.34-0.210.06-1.61-1.38-2.37-1.38-1.55-1.15-1.75-1.77-1.24-1.42-0.11 -1.070.00-1.36-1.28-0.69-0.530.15-0.19-1.68-1.93-0.35-1.20-1.82-1.30-0.95-1.86-1.75-0.44-1.53-1.40-1.65-0.61-0.49-1.69-1.51-0.64-1.53-0.730.00-1.92-1.51-1.45-0.53-1.19-1.32-2.800.00-1.09-1.48START M0.00-1.57NaN-1.25-0.98-0.32NaNNaN0.17-0.22-1.94-1.28-1.28NaN-2.27-0.85-0.10-0.13-1.40-1.25-0.370.00-1.04-0.10-0.38-0.48-0.85-0.35-0.18-0.69-1.14-0.02-0.24-1.31-1.64-0.180.04-0.16NaN-0.650.00-0.24-0.11 -0.530.00-0.45-0.20-1.580.05-1.10NaN0.00-1.64-0.30-1.10-0.13-1.05NaN-2.67NaNI-0.12-0.340.27-0.49-1.870.00-0.20-0.190.28-0.14-1.46-0.23-0.10-1.66-0.32-1.49-1.56-0.41-0.95-1.77-0.44-0.27-1.98-0.48-0.18-1.12-0.69-0.610.02-1.69-0.250.03-0.18-0.74-3.29-0.57-0.420.00NaN-1.29-0.280.000.03-1.61-0.34-2.10-0.37-1.71-0.13-0.90-1.33-0.15-1.54-0.54-0.12-0.14-0.37-0.81-1.35-1.25L-0.33-0.950.24-1.04-1.60-0.44-0.60-0.490.14-0.23-1.62-1.16-0.43-1.65-0.54-0.99-1.13-0.72-1.85-1.47-1.17-0.64-0.34-0.860.00-0.57-0.47-0.58-0.34-1.22-0.600.00-0.22-1.47-2.12-0.25-0.32-0.21-0.64-0.82-0.23-0.17-0.16-0.90-0.27-1.54-0.39-1.690.00-0.40-1.150.10-1.750.00-0.81-0.64-0.22-0.72-1.57-1.48V-0.29NaN0.32-0.33-0.53-0.18-2.16-0.74-0.36-0.26-0.46-1.340.00-0.230.00-0.040.000.00-1.95-1.57-0.38-0.52-0.53-0.98-0.28-1.31-0.72-0.05-0.15-0.390.00-0.10-0.04-1.96-0.15-0.13-0.29-0.30-0.01-0.42-0.140.050.00-0.85-0.42-1.37-0.14-0.86-0.48-0.93-0.39-0.35-0.31-0.610.000.00-0.44-1.72-0.53NaNA-1.70-1.64-0.60-0.10-0.33-1.56-1.220.100.320.23-0.99-0.16-0.59-0.65-0.670.00-0.43-0.39-1.70-1.40-0.19-1.33-0.87-0.66-0.77-0.86-0.270.000.00-0.12-0.57-0.28-0.08-1.050.00-0.14-0.06-1.470.00-0.14-0.77-0.78-0.18-0.22-1.11 -0.63-0.05-0.56-0.71-0.30-0.91-1.23-1.09-1.76-0.44-0.42-1.39-0.85-1.55NaNG-1.36-0.08-1.200.00-0.78-2.230.190.280.28-0.06-0.53-0.55-0.900.00-1.08-0.68-0.95-0.42-1.43-1.04-0.66-1.87-1.40-0.77-0.27-0.58-0.26-0.17-0.06-0.17-0.80-0.51-0.10-0.88-0.31-0.22-0.43-1.39-0.17-0.19-1.30-1.78-0.180.00-1.75-0.87-0.150.00-1.36-1.290.00-1.76-0.38-1.39-1.10-0.54-1.89-2.34-0.56NaNCNaNNaN-0.36-0.180.10-0.70-0.18NaN0.34-1.28-0.810.08-0.780.00-0.49-0.01-0.93-0.68-3.64-0.17-0.61-0.74-1.94-0.37-0.11 -1.10-0.07NaN-0.21-0.18-0.46-0.30-0.27-1.20-0.41-0.11 -0.19-1.77-0.90-0.49-0.51-0.81-0.15-0.27-1.10-1.450.07-0.64-0.56-0.94-0.31-0.43-1.90-0.97-0.31-0.44-0.54-1.94-1.77-0.26S-1.71-0.29-0.290.16-1.51-1.090.000.000.00-0.20-1.24-0.22-1.84-0.62-1.80-0.37-2.05-1.72-0.90-1.24-0.19-1.59-0.17-0.37-0.11 -0.650.000.02-0.02-0.65-1.21-0.43-0.26-0.27-0.59-0.11 -0.17-0.75-0.21-0.12-1.06-1.01-0.26-0.30-0.88-0.83-0.16-0.94-0.91-0.16-0.62-1.70-1.53-1.27-1.81-1.57-0.45-0.82-1.53-1.05T-0.43-0.620.28-0.99-1.71-0.54-0.490.200.250.17-0.980.00-1.67-1.30-1.02-0.41-1.19-1.29-1.06-1.92-0.05-0.66-0.49-0.63-0.54-0.560.000.01-0.12-1.12-0.720.03-0.08-1.25-0.51-0.12-0.22-0.67-0.29-0.17-0.35-0.32-0.22-0.89-0.65-0.80-0.19-1.77-0.73-0.36-0.94-0.47-1.10-1.53-1.03-1.33-1.48-0.62-2.18-0.83NNaNNaN-0.07-1.18-0.44-0.49-0.46NaN0.160.00-0.50-0.25-2.41-1.21-2.14-1.42-0.54-1.860.00-1.12-0.58-1.64-3.52-0.87-0.59-0.240.05-0.09-0.29-1.16-2.03-0.29-0.170.00-1.500.00-0.09-1.00-1.11 -0.03-1.52-0.41-0.13-0.24-1.83-0.42-0.26-0.62-1.11 -0.81-1.56-2.52-0.55-2.47-1.50NaN-1.57-1.81-1.69-0.69Q-1.67-1.420.31-1.10-0.94-1.67-1.34-1.040.340.17-1.16-0.67-2.35-1.19-1.96-1.37-1.58-1.89-0.38-1.86-0.47-2.05-0.34-0.72-0.33-0.18-0.510.07-0.12-0.06-1.92-0.050.00-0.69-2.37-0.20-0.05-1.00-0.54-0.24-0.16-2.070.01-0.48-1.32-0.530.00-2.43-0.84-0.53-1.36-1.74-1.70-0.72-1.18-1.57-1.43-2.87-0.97-0.86DNaN-0.67-0.59-0.200.00-1.57-1.80-0.900.26-0.140.00-1.70-2.08-0.16-1.84-0.43-2.11 -1.85-0.70-0.68-1.23-2.13-1.26-1.35-1.51-0.02-0.43-0.37-0.08-0.06-0.57-1.610.00-0.61-0.60-0.63-0.31-2.01-2.62-0.01-0.53-1.86-0.32-0.48-1.90-1.93-0.39-0.69-1.89-2.32-0.17-1.290.00-2.77-0.37NaN-1.42-2.05-0.50-0.27E-1.55NaN-1.10-1.01-0.46-1.57-1.20-0.90-0.41-0.37-0.42-0.36-0.49-1.42-0.58-1.72-0.49-0.49-1.84-2.42-0.18-1.59-1.46-1.64-1.41-0.61-0.59-0.43-0.060.00-1.69-0.260.06-0.27-1.00-0.24-0.13-2.02-0.140.00NaN-1.75-0.06-0.93-1.49-0.29-0.31-1.62-3.33-1.83-2.32-1.86-0.19-3.19-3.13-0.57NaNNaN0.00NaNH-5.77NaN0.00NaN-0.74-1.44-1.26-3.030.360.06-0.74-1.00-5.60NaN-1.11 -2.02-1.59-0.57-1.00-0.60-0.79-1.93-1.08-0.37-0.980.00-0.310.02-0.18-0.57-1.89-0.30-0.20-0.50-1.780.04-0.23-1.54-1.34-0.25-0.50-1.88-0.11 -0.23-0.90-1.33-0.16-1.12-0.74-0.97-1.70-1.64-1.18-0.51-1.50-2.11 -1.50-0.24-1.70-0.30K-0.44-0.47-1.01-2.37-1.69-1.69-2.180.30-0.630.15-1.66-1.15-2.30-1.92-1.57-1.38-1.67-1.43-0.46-1.410.00-0.87-0.31-0.57-1.58-0.52-0.240.28-0.16-0.58-1.57-0.02-0.29-0.53-1.81-0.050.00-1.28NaN-0.22-0.58-1.70-0.26-0.15-0.920.00-0.07-1.32-1.07-0.72-0.88-1.13-1.54NaNNaN-1.70NaNNaN-0.49-1.83R-0.690.00-0.56-0.86-1.39-0.92-0.23-0.39-1.380.11 -1.23-2.34-1.62-0.76-1.55-2.01-1.68-1.75-1.79-1.54-0.16-1.46-0.940.00-1.73-0.45-0.360.27-0.16-1.16-1.69-0.03-0.24-1.76-2.000.00-0.08-1.81-1.34-0.19-0.63-1.60-0.41-0.59-1.37-0.41-0.07-1.28-1.17-0.80-1.06-0.93-1.74-1.08-2.14-1.99-2.16-1.04-2.40-4.43AmiE WT 616263646566676869707172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110 111112 113 114 115 116 117 118 119 120Mutation SLQGIMYDPAEMMETAVAIPGEETEIFSRACRKANVWGVFSLTGERHEEHPRKAPYNTLVSTOP 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0.00-0.60-1.07-1.34-1.06-2.09-1.59-0.81-1.82-1.43-0.37-0.83-1.170.08-1.60-1.60-0.44-2.030.010.00-1.55-1.65-1.67-1.67-0.14-2.00-0.25-0.91-0.93-1.51-0.30-2.07-1.31NaN-1.75-1.77-1.45-1.76-1.45-2.65-1.33-2.26-0.17-1.09-2.95-2.37-4.09-2.74-3.69NaN-1.99DNaN-1.890.00-0.20-0.11 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WT 181182183184185186187188189190191192193194195196197198199200201202203204205206207208209210211 212213214215216217218219220221222223224225226227228229230231232233234235236237238239240Mutation KGAELIVRCQGYMYPAKDQQVMMAKAMAWANNCYVAVANAAGFDGVYSYFGHSAIIGFDGSTOP *-0.53-2.00-3.13-0.25-0.95-2.92NaN-2.51-0.69-0.50NaN-0.50-2.32-0.16NaN-2.33-1.01-1.37-0.58-0.53NaN-2.47-2.58-1.79-0.71-1.25NaN-2.90-0.08-2.98-3.36-3.84-0.43-0.39-2.59NaN-2.08-1.73-4.45-1.78-1.95-1.99-2.53-2.22-2.89-3.05-0.39-1.25-0.71-2.94####-2.52-2.45-1.64-3.77-2.99-2.86-2.67NaN-3.58F-0.93-2.33-5.53-2.74-0.12-0.50-0.63-2.70-0.46-3.62-1.68-0.05-2.33-0.61NaN-3.19-8.26-1.16-2.56-3.27-0.50-1.47-1.33-2.45NaN-3.16-1.79-1.57-0.58-2.07-3.84-1.00-0.28-0.66-0.65-1.97-2.11 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M0.00-2.15-1.78-3.88-0.49-0.45-2.86NaN-3.480.20NaNNaN0.00NaNNaN-3.29-1.32-1.66-0.54-3.530.370.000.00-2.21-0.17NaN0.00-2.09NaN-1.75NaNNaNNaNNaNNaNNaN-1.38-1.96-0.97-1.89NaNNaN-1.59NaNNaN-0.54-3.62NaN-2.59NaN-0.72NaN-3.970.33-0.57-0.72NaN-1.23-1.75NaNINaNNaN-2.09NaN-1.310.00-0.13-7.44-2.39-2.40-2.33-2.10-0.24-1.79NaN-2.13-0.67-1.41-0.53-0.860.20-0.06-0.44-1.81-2.93-1.41-0.28-2.11 NaNNaN-0.14-0.49-0.99-2.30-0.63NaN-0.40-3.02-0.87-0.06-2.15-2.52-1.45-2.98-1.80-0.15-2.74-0.20-2.61-0.54NaN-3.10-0.89-0.460.000.00NaN-0.79-4.00-1.66L-2.36NaN-2.00-2.220.00-0.31-1.54-0.50-2.39-0.54-2.13-2.25-0.39-2.26-0.43-3.31-1.20-0.69-0.62-1.11 0.16-0.03-0.62-2.63-1.31-1.90-0.31-2.08-0.35-2.35-3.09-0.83-1.17-2.67-1.81-2.13-0.65-1.87-3.15-0.72-3.54-2.66-0.73NaN-2.10-0.94-1.63-6.36-3.32-0.25-3.96-0.48-2.48-0.18-0.66-0.42-2.66-0.53-4.16-3.96V-2.16-0.52-0.10-0.45-0.35-0.160.00NaN-1.33-3.11 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-0.64-0.83-0.42-0.67-0.32-1.57-2.33-0.24-2.21-2.190.00-2.75-5.15-0.67-2.02-0.71-0.70G-1.780.00-1.27-0.56-2.51-2.35-0.50-0.58-1.05-1.310.00-3.59-0.85-1.92NaN-0.44NaN-0.23NaN-1.99-0.18-0.72-2.25-0.41-2.32-0.87-1.75-0.93-0.90-0.61NaN-0.27-0.56-2.68-0.95-0.49-1.07-0.61-1.96-0.37-0.480.00-3.73-0.320.00-0.36NaN0.05-3.54-2.210.00-0.91-0.53-0.76-3.31-2.740.00-2.99-0.120.00C-2.95-0.42-1.36-4.26-1.35-1.91-0.13-0.170.00-0.30-0.53-0.54-1.86-0.12-1.43-0.30-1.20-1.22-1.25-1.090.13-0.42-2.470.23-2.210.15-2.09-1.44-0.01-0.46-1.75-0.370.00-0.69-1.64NaN-0.43-1.40-3.25-0.71-0.30-0.31-0.48-4.43-0.15-0.12-0.17-0.20-0.20-0.18-0.14-0.68-0.610.28-3.38-2.89-0.53-0.60-1.33-0.32S-1.98-1.26-0.39-5.84-0.58-1.11 NaN-0.740.04-0.04-0.42-0.95-1.54-0.37-0.97-0.25-1.58-1.23-1.03-0.140.02-0.48-2.790.10-1.39-0.21-3.84-0.66-0.90-0.68-0.61-0.33-0.62-1.91-2.66-0.32-1.82-0.54-0.37-0.55-0.02-1.30-0.85-1.71-0.47-1.03-0.590.00-1.14-0.23-0.26-1.990.00-0.04-1.48-0.710.08-0.88-3.47-0.48T-1.18-2.30-0.48-2.64-2.99-0.80-2.96-2.35-0.77-0.49-3.20-2.36-0.38-2.36-0.68-0.26-0.89-0.32-0.39-1.840.20-0.11 -0.81-0.17-0.860.02-0.64-0.64NaN-0.44-1.48-0.25-0.51-2.29-2.61-0.18-0.46-0.12-0.91-0.510.04-3.09-0.81-2.68-3.02-0.34-3.06-0.37-2.90-0.78-1.78-1.60-1.620.07-0.85-0.78-2.68-3.10-2.15-2.80N-0.38-2.70-1.08-2.21NaN-0.23-3.48-2.54-1.35-0.02NaN-1.09-1.77-0.10-1.11 -0.84-0.43-0.46-1.33-1.21-0.11 -0.12-3.78-0.28-0.57-1.25-2.86-2.23-2.09-2.390.000.00-1.60-1.04-2.71-3.22NaN-4.120.00-2.34NaNNaN-0.97-0.48-2.80-0.59-0.57-0.69-0.63-2.36-2.02-0.13-0.71-1.55-0.74-0.66-1.11 -3.22-0.83-2.23Q-0.66-2.02-3.44-1.26-2.26NaN-2.05-1.53-1.800.00NaN-3.38-2.03-0.15-2.99-1.02-0.24-0.600.000.000.01-0.35-2.26-1.50-0.62-1.80-1.58-3.13NaN-3.08-2.440.00-2.04-2.10NaN-3.01-0.08-5.05-2.97-3.92-2.74-2.83-0.20-2.19NaN-1.24-1.29-1.73-3.19NaN-3.86-0.10-3.440.06-3.19-1.96-4.92-1.95-2.55-2.78DNaN-0.54-2.15-0.37-1.88-0.81-0.19-1.67-1.23-3.29-0.15-0.44NaN-0.19NaN-0.22-2.610.00-1.11 -2.52-0.51-1.95NaN-0.26NaN-1.50-2.43-0.73-1.49-2.15-0.54-0.25-2.49-0.68-0.49-0.25-3.66NaN-0.38-0.28-2.35-0.42-2.380.00-0.52-0.81-0.35-2.76-0.13-2.02-0.54-0.45-2.26-0.81NaNNaN-0.43NaN0.00-0.32E-0.27-2.95-0.560.00-2.82-3.29-2.09-1.89NaN-0.35NaN-2.68-2.12-3.62NaNNaN-0.28-0.01-0.73-0.80-0.74-1.67NaN-2.15-0.33-0.88-3.29-2.06-2.15-0.23-1.82-1.03-2.77-2.98-2.96-2.09-0.82-0.22-3.55-1.73-0.27-4.04-2.05-0.22-2.22NaNNaN-1.92-1.82NaNNaN-2.02-3.31-2.06-1.98-2.12-3.62-1.41-0.57-1.96H-1.38-3.27-2.48-2.27-4.25-1.64-5.01-0.41NaN-0.19-1.75-0.21-5.73-0.53-0.20-3.16-1.63-0.61-0.10-0.32-0.64-0.08-2.12-1.61-1.13-2.11 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-1.31-2.51-2.22-0.63-0.43-3.42-1.37-0.25-2.06-4.95-0.15-0.11 0.22-1.92-0.23-0.77-0.16-0.15-0.43-0.49-0.20-0.380.07-0.07-0.250.18-0.24-0.20KNaN-2.24-0.71-2.35-1.140.09-1.21-1.14-1.19-0.13-1.42-1.69-0.090.00-1.89-1.00-0.21-0.40-1.97-0.470.15-2.60NaN-0.430.16-0.09-0.15-0.35-0.930.06-0.06-0.17-0.11 -0.47-0.120.21-0.18-0.390.03-0.120.04R-1.29-2.50-2.44-2.91NaN0.00-1.40-0.11 -2.21-0.31-2.48-1.84-0.89-0.13-2.490.00-0.34-0.54-2.27-0.830.00-0.79-2.770.00-0.06-0.24-0.75-0.22-1.04-0.02-0.34-0.41-0.26-0.97-0.310.00-0.18-0.37-0.04-0.15-0.13Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged ! 120 Figure A 19: AmiE I122L heatmap. AmiE I122L 123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960Mutation MRHGDISSSNDTVGVAVVNYKMPRLHTAAEVLDNARKIAEMIVGMKQGLPGMDLVVFPEYSTOP *-1.72-0.23-1.09-1.48-1.36-1.51NaN-0.20-0.16-1.44-1.61-2.35-1.34-1.10-1.59-1.47-1.78-1.43-1.39-0.52-0.64-1.86-1.34-1.52-1.11 -1.51-1.67-1.64-1.50-0.30-1.40-1.27-1.16-1.48-2.08-0.93-0.72-1.59NaN-0.57-1.67-1.23-1.48-2.47-1.55-0.51-0.71-1.70-1.42-1.73-1.74-1.56-1.62-1.11 -1.82-1.29-1.03NaN-0.25-0.10FNaNNaN-0.46NaN-1.64-0.46NaN-1.220.21-1.32-1.15-1.35NaN-1.35-1.35-1.79-1.59-1.39-1.87-0.29-1.80-1.48-1.12-1.40-0.25-0.88-0.93-1.23-0.60-1.27-0.72-0.68-0.68-1.42-1.66-0.80-0.60-0.50NaN-1.59-0.20-0.54-0.60-0.75-0.92-1.41-0.77-1.23-0.04-1.36-1.21-1.43-1.06-1.14-0.30-1.930.00-1.17-1.23-0.08WNaNNaNNaNNaN-1.77NaNNaN-0.21NaN-1.33-1.12NaN-1.09-0.85-1.64-0.95-1.14-1.44-1.15-0.61-1.89-3.09-1.39-3.41-1.70-0.81-1.82-1.22-0.64-1.70-1.08-1.65-0.54-1.80-1.31NaN-1.17-1.00-1.17-1.45-1.17-1.73-0.53-0.67-1.42-1.26-0.98-1.52-0.50-1.10-1.13-1.33-1.22-2.13NaNNaNNaNNaN-1.43NaNY-0.93NaN-0.23-1.87-0.57-1.45NaN-1.130.34-0.46-0.33-1.11 -1.76-2.08-1.83-1.41-1.50-2.18-0.460.00-1.49-1.23-1.71-1.08-1.20-0.18-0.78-0.75-0.52-1.18-1.28-0.72-0.32-1.11 -2.67-0.36-0.45-4.79NaN-1.06-0.93-1.36-0.34-0.74-1.46-1.76-0.59-0.94-0.01-1.12-1.47-1.60-0.54-1.55-1.79NaN-0.20NaN-1.520.00P-1.84-1.380.41-1.61-1.72-1.73NaN-0.57-0.360.03-1.65-0.93-1.46-1.22-1.28-1.19-1.49-1.54-1.89-1.35-0.10-0.980.00-1.23-1.32-0.69-1.040.10-0.35-1.28-1.43-0.32-1.21-2.09-0.74-0.56-1.34-1.38-0.38-1.73-1.34-1.69-0.87-1.14-1.47-1.39-1.00-1.67-0.390.00-1.56-1.36-1.59-0.65-1.22-1.43NaN0.00-1.54-2.35START M0.00NaNNaN-1.22-1.73-0.58NaN-1.440.06-0.27-2.54-0.66-1.00NaN-1.32-1.85-0.30-0.26-1.27-1.64-0.210.00-0.79-1.43-0.38NaN-0.82-0.35-0.23-0.49-1.430.03-0.29-1.35NaNNaN-0.41-0.97NaN-0.720.00-0.42-0.22-0.550.00-0.29-0.28-1.400.05-1.25-1.950.00-1.67-0.29-1.22-0.05NaN-0.85NaNNaNI-0.19-0.200.23-0.93-1.490.00-0.010.070.27-0.21-1.30-0.20-0.17-2.30-0.39-1.43-1.60-0.40-0.65-1.29-1.25-0.26-2.12-1.10-0.11 -1.14-0.72-0.74-0.35-1.63-0.260.09-0.40-0.78-1.60-0.85-0.430.00-1.06-1.65-0.150.000.03-1.85-0.31-1.67-0.59-1.42-0.13-1.04-1.50-0.16-1.60-0.78-0.19-0.03-0.15-1.47-1.12-0.40L-0.32-2.060.20-1.86-1.57-0.59NaN-0.36-0.14-0.26-1.64-1.34-0.45-1.33-0.61-1.29-1.12-0.75-1.46-1.45-1.13-0.69-0.37-1.010.00-0.56-0.92-0.68-0.43-1.17-1.140.00-0.33-1.53-1.26-0.30-0.51-0.64-1.50-1.15-0.25-0.40-0.30-0.79-0.09-1.87-0.51-1.440.00-0.53-1.830.24-1.420.00-0.59-0.45-0.06-0.28-1.44NaNV-0.29-1.270.16-0.29-0.59-0.13-1.22-0.36-0.38-0.35-0.58-1.530.00-0.330.00-0.130.000.00-1.59-1.45-0.66-0.61-0.98-0.75-0.17-1.31-1.03-0.12-0.25-0.380.00-0.16-0.16-1.69-0.15-0.59-0.48-0.25-0.10-0.62-0.250.090.00-0.65-0.45-1.71-0.39-1.02-0.52-0.95-0.66-0.37-0.56-0.840.000.00-0.23NaN-0.32NaNA-1.13NaN-0.270.08-0.78-1.45-0.660.050.180.17-1.25-0.16-0.33-0.54-0.540.00-0.56-0.61-1.55-1.22-0.28-1.73-0.97-0.78-0.52-1.05-0.390.000.00-1.01-0.62-0.43-0.05-1.180.00-0.20-0.15-1.620.00-0.13-1.26-1.12-0.33-0.10-1.08-0.89-0.10-0.39-1.13-0.75-1.23-1.07-1.82-1.82-0.26-0.31-1.24-0.42-1.22-1.22G-1.57-0.42-1.420.00-0.54-1.24-0.070.270.26-0.11 -0.52-0.60-0.750.00-0.83-0.96-0.85-0.48-1.32-1.67-0.59-2.43-1.91-0.93-1.33-1.23-0.28-0.09-0.04-0.30-0.81-0.72-0.15-1.08-0.60-0.29-0.56-1.64-0.06-0.21-1.12-1.92-0.420.00-1.80-2.05-0.600.00NaN-1.510.00-1.70-0.66-1.31-0.53-0.51-0.96-1.35-0.24NaNC-1.31-1.31-0.22-0.05-0.20-1.12-0.11 0.230.20-1.52-1.18-0.17-0.98-0.13-0.85-0.12-0.92-0.51-1.80-0.28-0.60-1.13-1.77-0.39-0.04-1.04-0.19-0.09-0.23-0.67-0.68-0.37-0.33-1.64-0.92-0.13-0.24-1.13NaN-0.62-0.63-0.82-0.15-0.17-1.19-1.36-0.44-0.55-0.62-1.01-0.55-0.40-1.88-1.97-0.63-0.54-0.33NaN-1.10-0.09S-1.35-0.430.040.06-1.48-0.990.000.000.00-0.22-1.42-0.20-1.47-0.38-1.41-0.48-1.55-1.41-0.74-1.71-0.26-1.42-0.22-0.41-0.50-0.530.040.120.04-0.56-1.40-0.68-0.15-0.46-0.43-0.08-0.21-0.88-0.15-0.14-1.30-0.91-0.45-0.14-1.54-1.06-0.20-1.10-1.28-0.27-0.90-2.26-1.44-1.65-1.28-1.23-0.20-0.22-1.71-0.84T-0.37-0.800.04-0.90-1.55-0.63-0.180.140.20-0.02-2.020.00-1.95-2.03-1.31-0.48-1.31-1.26-1.27-1.52-0.22-0.67-0.72-0.63-0.47-0.820.000.01-0.08-0.97-0.74-0.22-0.20-0.40-0.36-0.18-0.51-0.62-0.16-0.30-0.30-0.52-0.32-0.67-0.81-1.08-0.23-1.60-0.73-0.75-1.65-0.56-1.54-1.34-2.03-1.71-1.26-0.11 -1.37-2.06N-1.47-2.010.07-0.47-0.34-0.64-0.100.030.170.00-0.50-0.10-1.83-1.52-1.66-1.69-1.54-1.220.00-1.06-0.65-1.40-1.35-0.77-0.62-0.490.000.02-0.14-0.91-1.25-0.59-0.11 0.00-1.560.13-0.13-0.60NaN-0.01-1.90-0.60-0.52-0.12-1.37-0.55-0.29-1.53-1.62-1.39-1.75-2.42-0.53-1.36-2.13-1.00-1.57NaN-2.13-0.08Q-1.27-1.59-0.07-1.41-1.45-1.78NaN-1.120.120.19-1.51-0.62-2.37NaN-2.17-1.34-1.47-3.36-0.60-1.51-0.65-1.40-0.34-0.65-1.18-0.86-0.730.22-0.06-0.02-1.38-0.15-0.06-0.60-1.33-0.11 -0.22-1.77NaN-0.16-0.50-1.31-0.09-0.36-1.37-1.030.00-1.60-0.61-0.49-1.69-2.10-1.73-0.59-1.54NaNNaNNaN-0.29NaND-1.57-2.16-0.48-0.220.00-1.61NaN-0.850.41-0.060.00-1.90-1.11 -0.23-1.31-0.71-1.34-1.40-0.50-0.95-1.27-1.64-1.93-1.24-1.37-0.70-0.49-0.30-0.03-0.71-0.68-1.760.00-0.53-0.35-0.75-0.55-2.13-0.930.22NaN-3.10-0.42-0.43-1.93-2.17-0.65-0.82-1.50-1.37-0.28-1.320.00-2.06-0.17-1.90-0.96NaN-0.44-0.79E-1.38-1.76NaN-0.85-0.52-1.56NaN-1.13-0.21-0.12-0.46NaN-0.22-1.22-0.50-1.25-0.51-0.52-1.29-1.74-0.21-1.19-3.34-1.38-1.37NaN-0.82-0.52-0.090.00-1.44-0.800.00-0.73-1.85-0.21-0.23-1.35-0.080.00-1.39-1.82-0.10-0.71-1.50-0.37-0.35-1.88-2.08-1.17-2.37-1.44-0.22-1.46NaN-0.13NaN-0.850.00NaNHNaNNaN0.00-1.71-1.17-2.36NaN-1.550.26-0.26-0.85-1.16-1.55-1.17-1.80-1.52-2.18-2.21-1.11 -0.61-0.92-1.25-1.50-0.44-1.050.00-0.390.09-0.17-0.57-1.53-0.37-0.27-1.23-1.480.00-0.17-1.85-1.00-0.21-0.90-1.79-0.25-0.20-1.67-1.57-0.19-1.31-0.84-1.21-1.79-1.47-1.23-1.24-1.54-0.70-1.700.13NaN-0.46K-0.51-0.38-0.85NaN-1.44-1.34NaN0.14-1.000.03-1.07-1.47-1.26-1.45-1.64-1.72-1.47-1.42-0.50-1.750.00-0.90-1.31-0.36-1.45-0.46-0.310.43-0.07-0.32-1.730.02-0.30-0.58-1.980.120.00-1.52NaN-0.21-0.24-1.88-0.50-0.63-0.740.000.07-1.51-1.14-1.23-2.41-0.65-2.17-2.79NaNNaN-0.85NaN-0.17-1.43R-0.750.00-0.21-0.84-1.29-1.280.00-0.40-1.450.02-1.38-1.49-1.64-0.54-1.36-1.55-1.51-1.28-1.36-1.33-0.17-1.38-0.940.00-1.46-0.24-0.340.36-0.13-1.06-1.240.04-0.29-1.54-1.620.00-0.07-1.43-1.76-0.20-0.62-1.44-0.58-0.37-1.35-0.570.03-1.39-0.80-0.99-1.26-0.35-1.67-1.25-1.26-3.39-1.06-0.46-1.16-1.40AmiE I122L 616263646566676869707172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110 111112 113 114 115 116 117 118 119 120Mutation SLQGIMYDPAEMMETAVAIPGEETEIFSRACRKANVWGVFSLTGERHEEHPRKAPYNTLVSTOP *-1.17-1.03-0.49NaN-1.98-1.57-0.48-1.72-1.56-1.43-0.35-1.46-1.37-0.66-1.30-1.82-1.26-1.63-1.44-1.85-1.60-0.76-0.57-1.30-0.13-1.67-1.60-1.35-1.92-1.68-0.27-0.77-0.71-1.72-2.05-1.76-0.54-2.32-1.75-1.81-1.83-1.95-1.87-2.23-0.83-1.31-1.62-0.63-0.28-1.35-1.53-1.00-0.61-2.33NaNNaNNaNNaNNaNNaNF-0.18-0.36-1.55NaN-0.44-1.35-0.17-1.30-0.49-0.40-0.49-1.02-0.26-1.31-0.59-1.32-0.96-0.57-0.76-1.53-1.98-0.68-1.38-1.97-0.73-0.620.00-1.00-0.69-1.73-0.31-1.60-1.27-1.63-3.66-0.79-0.93-1.78-1.470.00-0.33-0.25-2.26-1.59-1.64-1.12-1.58-1.82-0.66-1.26-1.68-0.18-2.02-0.90NaNNaNNaNNaNNaNNaNWNaN-1.00-2.67-0.85-1.33NaN-0.66-1.30-0.38-0.44-0.76-1.24-0.72-1.56-1.22-1.32NaN-0.51-1.64-1.22-1.45-0.99-1.51-1.37-1.54-0.84-0.37NaN-0.06NaN-0.17-1.50-1.45-2.74NaN-1.200.00-1.50NaN-1.17NaN-1.81-3.53-1.80-2.07NaNNaN-1.71NaN-1.24NaN-0.59NaNNaNNaNNaNNaNNaNNaNNaNY-0.11 -1.70-2.15NaN-1.47-1.790.00-0.51-0.27-0.25-0.84-1.19-0.08-0.76-0.64-2.61NaN-0.25-1.38-1.20-1.57-0.50-1.57-1.49-1.00-1.52-0.31NaN-0.26-1.31-0.23-1.29-0.78-1.76-0.42-2.66-0.92-2.15NaN-0.51-0.77-1.30-1.73-1.75-1.59NaN-0.41-2.98-0.70-0.42-2.44-0.01-1.49-0.74NaNNaNNaNNaNNaNNaNP-0.26-1.55-1.09-1.53-0.97-0.43-1.05-1.440.00-0.82-1.09-1.73-1.87-1.27-1.15-1.00-1.24-0.17-1.530.00-1.620.34-1.33-1.38-1.54-1.95-1.91-1.33-0.89-1.01-1.35-1.62-1.18-1.50-1.89-2.15-1.91-2.11 -2.30-1.88-0.75-0.53-1.57-1.97-2.33-0.56-1.47-0.61-1.84-1.410.00-2.16-1.52-0.49NaNNaNNaNNaNNaNNaNSTART MNaN-0.76-1.45NaN-0.860.00-0.73-1.11 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I122L 121122123124125126127128129130131132133134135136137138139140141142143144145146147148149150151152153154155156157158159160161162163164165166167168169170171172173174175176177178179180Mutation LLDNNGEIVQKYRKIIPWCPIEGWYPGGQTYVSEGPKGMKISLIICDDGNYPEIWRDCAMSTOP 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-0.07-2.93-0.42-0.27-0.59-0.29-0.77-1.73-2.05-0.20NaN-1.02-1.15-1.68-0.41-1.190.390.00-1.18NaN-0.48-1.20-0.18-1.25NaN-0.87-0.07-1.69-1.330.00-1.13-1.65SNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.81NaN-0.88-1.73-1.53-0.39-0.86-1.41-0.54-0.23-0.69-2.150.06-0.29-1.00-0.60-0.940.10-0.09-0.92-1.48-1.840.00-0.18-1.33-0.35-2.05-0.31-1.59-1.460.050.00-0.65-0.86-0.74-0.80-1.41-1.76-0.54-0.520.40-0.47-1.44-0.91-0.53-0.41-1.89-0.24-0.47-1.19TNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.07NaN-0.95-1.50-0.77-0.87-0.69-1.610.01-0.35-0.40-2.22-0.04NaN-0.56-0.73-1.850.14-0.100.00-1.45-1.40-0.050.05-1.61-0.53-2.79-1.24-0.22-1.20-0.65-0.93-1.43-0.73-0.18-2.15-1.47-2.25-2.19-1.04-1.42-0.25-2.39-0.25-2.29-1.26NaN-1.27-0.26-0.45NNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.97NaN-2.42-0.63-0.62-0.14-1.71-3.33-1.18NaN-0.54-2.630.03NaN-0.58-1.59-1.220.25-0.03-2.22-0.96-2.26-0.71-0.04-1.91NaN-0.31NaN-1.09-0.76-0.81-2.29-1.66-0.41-0.45-2.67-0.31-0.50-1.680.00-0.72-1.37-1.06-0.54NaN-1.34-0.44NaN-1.88-1.83QNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.89NaN-1.51-1.16-1.39-1.46-0.54-1.73-2.02-0.21-0.64-0.960.14NaN-2.96-0.44-2.080.010.00-1.90-1.95-1.76-2.57-0.16-1.99-0.23NaNNaNNaN-0.72-1.33-1.93-1.77NaN-1.50-1.73-1.70NaN-1.91-2.07-2.76-0.31-0.60-0.97-1.24-1.58-2.62-0.81-1.57-1.87DNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-3.46NaN-1.33-2.40-2.65-1.94-1.99-2.88-0.58NaN-2.80-0.43-0.45NaN-0.97-1.97-0.680.00-0.55-2.24-1.22-0.80-1.090.05-0.71NaN-2.05-0.12NaN-1.78-1.59-1.47-3.00-1.31-1.88-2.270.000.00-0.69-0.24-0.77-1.70-0.37-1.05-1.07-1.830.00-0.76-0.21-1.54ENaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.60NaN-1.78-0.32-1.27-1.94-1.96-2.08-1.51NaN-3.190.00-0.45NaN-4.06-2.31-1.950.07-0.10-2.25-1.47-1.71-7.100.00-2.47-1.31-0.41NaNNaN-0.44-1.60-1.75-1.85NaN-1.57NaN-1.87-0.40-1.68-0.87-2.23-1.030.00-1.42NaN-1.12-0.20NaN-2.67-1.85HNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.82NaN-1.03-1.71-1.92-0.22-1.80-1.52-1.62-1.95-0.52-2.460.01NaN-0.48-1.93-1.62-0.03-0.16-1.68-0.48-2.13-2.12-0.29-1.83-2.17-2.11 -1.60-0.75-1.21-1.49-2.14-2.07-1.29-1.30-1.83-0.85-0.68-2.21-1.49-0.29-1.46-1.19NaN-1.49-2.00-0.53-1.19NaN-2.95KNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN0.00NaN-0.550.00-3.05-2.39-1.53-2.04-3.58-1.22-0.67-0.64-0.18NaN-1.71-2.07-1.400.11 -0.16-1.03-2.54-1.64-1.71-0.22-1.62-1.810.00-1.31-0.190.00-2.26-1.91-1.90-2.02-1.67-1.67NaN-1.57-2.01-0.13-1.15-1.12-0.23-2.62-1.78-0.72NaN-1.22-1.50-0.53RNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.15NaN0.00-0.70-1.82-1.72-1.30-0.83-0.77-0.36-1.08-2.02-0.13-0.10-2.16-0.84-1.92-0.09-0.36-1.32-1.88-1.70-1.79-0.27-1.72-0.77-0.53-0.63-0.68-0.26-1.82-1.71-1.70-2.60-2.29-0.72-2.09-3.85-1.38-2.39-1.45-1.03-1.70-1.78-0.270.00-1.70-0.18-1.69-0.54AmiE I122L 181182183184185186187188189190191192193194195196197198199200201202203204205206207208209210211 212213214215216217218219220221222223224225226227228229230231232233234235236237238239240Mutation KGAELIVRCQGYMYPAKDQQVMMAKAMAWANNCYVAVANAAGFDGVYSYFGHSAIIGFDGSTOP *-0.59-1.59-1.76-0.29-1.05-1.34-1.25-2.24-0.52-0.08-1.00-0.54-1.95-0.25NaN-2.82-0.39-1.71-0.35-0.77-2.48-1.85-1.57-3.25-0.56-2.26-1.49-1.74-0.15-1.41-2.77-1.46-0.35-0.48-1.80-1.21-1.59NaNNaN-0.84-2.20-1.99-1.58-1.32-1.54-2.14-1.00-1.42-0.30-2.11 NaN-1.86-1.54-1.57-1.55-1.57-1.80-1.77NaN-1.40F-1.27-1.44-1.57NaN-0.50-0.26-0.55-1.15-0.34-1.11 -1.76-0.08-2.19-0.39-1.63-1.34-1.55-0.68-1.37-1.52-0.46-0.90-1.27-2.31-1.46-1.84-1.51NaN-0.46-1.52-1.81-1.21-0.31-0.43-0.59-1.70-1.59-1.46-1.49NaN-1.65-2.440.00-1.49-1.43-0.21-0.03NaN-0.040.00NaN-1.25-2.21-1.33-0.68-0.26-1.270.00-1.15-1.39W-1.45-2.57-1.25NaN-0.79-1.41-2.05NaN-0.44NaN-1.38-1.56-1.69-1.38-1.29NaN-0.62-1.16NaN-1.99-0.70-0.56-1.24NaNNaN-0.96-1.98NaN0.00-2.02NaN-1.46-0.68NaN-1.30NaN-1.61-1.15-0.98NaNNaN-1.42-0.28-0.84NaNNaNNaNNaNNaN-0.34NaNNaN-1.02-1.35-2.00-1.83-1.49NaNNaN-1.00Y-1.73-1.67-1.47-1.30-1.97-1.46-2.18-1.65-0.10NaN-1.320.00-1.050.00-0.98-1.70NaN-0.38-1.23-2.30-0.63-0.87-1.46-2.54-1.54-2.94-1.30NaN-0.54-1.47-0.41-0.61-0.350.00-1.50-1.76-2.17-2.38-0.35NaN-1.00-1.74-0.18-0.25NaN-0.960.00-0.910.00-0.03NaN-0.17-1.58-1.88-1.650.21-2.26-0.39-0.24-1.90P-1.51-1.66-1.31-2.34-1.80-1.61-1.50-0.92-0.92-0.46-1.82-1.37-0.08-1.600.00-0.89-1.87-0.88-0.41-1.06-1.61-1.40-1.60-0.75-1.10-0.79-2.06-0.59-1.76-1.16-1.83-1.38-1.49-1.72-1.67-0.93-1.79-0.76-1.15-0.54-0.79-1.51-1.61-1.47-1.71-1.20-2.19-1.51-1.16-1.23-1.12-0.88-1.61-1.17-1.83-1.61-1.59-1.60-1.48-1.70START M-0.44-1.59-2.00NaN-0.69-0.44-1.83NaN-1.51NaN-0.93-1.570.00-1.15NaN-1.17-0.40-0.76-0.58-1.280.310.000.00-1.41-0.01-1.190.00-0.98-1.30-1.97-1.94-0.37-1.06-1.40-1.17NaN-1.50NaN-1.13NaNNaN-1.20-1.23NaNNaN-0.66-0.74-1.07NaN-0.74NaN-0.95-2.260.19-6.33-0.80-2.21-1.03NaN-1.86I-1.63-1.53-3.68-1.76-0.950.00-0.06NaN-1.34-1.54-2.02-2.10-0.35-1.82-1.08-1.72-0.45-0.98-0.36-1.500.20-0.07-0.26-1.53-1.35-0.88-0.19-2.02-1.11 -1.20-0.45-0.37-0.86-1.25-0.40-1.56-0.33-1.58-0.39-0.81-1.44-1.56-1.19-1.97NaN-0.10-1.82-0.33-1.61-0.52NaN-1.28-0.67-0.550.000.00-1.80-0.94-1.28-2.27L-1.47-1.72-1.78-1.610.00-0.90-0.94-0.47-1.75-0.18-1.76-1.80-0.66-1.31-0.36-1.34-0.91-0.96-0.54-1.180.17-0.08-0.30-1.75-1.76-1.52-0.66NaN-0.37-1.60-1.77-0.70-1.25-1.71-0.96-1.36-0.56-3.12-1.66-1.51-1.74-1.30-0.66-1.70-2.51-0.49-1.17-1.83-2.39-0.53-1.94-0.52-2.51-0.42-0.57-1.12-1.96-0.51-1.37-1.57V-1.68-0.83-0.19-0.21-0.93-0.270.00-1.46-1.29-1.11 -0.23-1.75-0.42-2.99-1.26-0.15-0.87-0.49-0.34-1.460.00-0.19-0.25-0.29-1.35-0.08-0.44-0.12-1.66-0.32-1.63-0.370.03-1.780.00-0.400.00-0.17-1.63-0.15-0.23-1.02-1.10-0.24-0.210.00-1.56NaN-0.86-0.78-0.63-3.13-1.43-0.34-0.53-0.52-0.57-0.53-0.09-0.44A-1.33-1.310.00-1.21-1.46-2.10-0.46-1.24-0.080.07-0.51-1.39-0.71-0.16-0.580.00-1.03-0.71-0.18-1.00-0.07-0.09-1.290.00-2.380.00-1.930.00-1.900.00-1.70-0.95-0.52-1.59-0.570.00-0.540.00-1.260.000.00-1.58-1.31-1.13-0.74-0.26-0.87-0.25-1.09-1.66-0.41-1.23-1.510.00-1.66-1.82-0.59-2.04-0.61-0.94G-1.000.00-1.10-0.27-1.53-1.70-0.70-0.41-0.81-0.960.00-1.35-0.47-1.93-2.07-0.34-1.60-0.63-1.43-1.46-0.16-0.38-1.09-0.61-1.86-0.61-1.51-0.36-0.83-1.05-1.47-0.62-0.50-1.82-0.86-0.83-0.98-0.69-2.78-0.24-0.640.00-1.52-0.540.000.02-1.32-0.16-1.78-2.070.00-0.65-0.97-0.65-1.51-1.530.00-1.54-0.160.00C-1.39-0.59-1.15-1.84-1.09-2.05-0.39-0.200.00-0.22-0.21-0.39-0.46-0.28-1.540.04-0.81-1.00-0.81-1.550.07-0.15-2.230.10-0.930.13-1.18-1.74-0.11 -0.32-3.78-0.330.00-0.32-1.46-0.81-0.17NaNNaNNaN-0.84-0.62-0.53-0.86-0.18-0.20-0.04-0.12-0.02-0.31-0.25-0.76-0.440.14-1.48-1.44-0.43-0.22-1.04-0.26S-1.21-1.14-0.64-1.68-0.78-0.82-1.64-0.500.01-0.06-0.37-1.32-0.78-0.16-0.37-0.18-0.57-0.58-0.77-1.180.02-0.40-1.970.16-1.14-0.08-2.07-0.35-0.71-0.83-0.69-0.31-0.35-1.60-1.93-0.61-1.03-0.29-0.48-0.200.00-1.24-0.62-1.45-0.22-0.65-0.840.00-1.34-0.15-0.05-1.430.00-0.04-3.08-0.99-0.19-0.52-1.84-0.44T-1.10-1.56-0.50-1.68-2.17-0.54-1.95-1.77-1.74-0.37-1.08-1.52-0.46-0.99-0.24-0.30-0.97-0.68-0.45-1.410.13-0.03-0.50-0.08-0.73-0.01-0.49-0.22-1.68-0.53-1.28-0.24-0.15-1.45-1.49-0.23-0.39-0.21-0.55-0.190.10-1.88-0.95-1.46-1.33-0.09-1.73-0.29-0.92-0.41-1.72-1.20-1.27-0.08-0.99-0.99-1.38-1.36-1.61-1.54N-0.56-1.55-2.63-1.19-1.82-0.35-1.79-1.45-1.93-0.66-1.90-0.80-0.85-0.09-1.49-0.48-0.47-0.40-1.29-0.76-0.07-0.01NaN-0.16-0.18-1.20-1.06-1.51-2.09-2.140.000.00-1.55-1.04-1.59-1.57-1.72-1.480.00NaN-1.89-1.59-0.47-0.35-1.82-0.97-0.53-0.28-0.26-1.92-0.84-0.17-0.68-1.21-0.78-0.75-1.03-1.77-0.23-1.27Q-0.67-1.49NaN-0.45-1.90-1.64-1.75-1.42-1.690.00-1.19-1.63-1.15-0.04NaN-0.86-0.48-0.550.000.000.09-0.29-1.12-0.76-0.74-1.56-1.43NaN-1.70-1.63-1.430.12NaN-1.73-1.82-1.75-0.34-1.56-1.30NaN-1.90-3.15-1.06-2.17-0.97-0.66-1.84-1.30-1.35NaNNaN-0.50-1.570.14-1.75-1.81-1.45-1.15NaN-1.60D-1.66-0.64-1.32-0.26-5.05-1.43-0.65-1.47-2.08-1.19-0.14-0.84-2.57-0.92-1.39-0.43-1.190.00-1.87-1.44-0.49-1.40-1.82-0.45-1.56-2.29-2.33-0.18-1.28-1.50-0.40-0.59-0.98-4.36-0.42-0.51-1.75-2.02-0.23-0.16-1.23-0.87-1.910.00-0.22-0.31-0.62-1.16-0.29-1.14-0.05-0.54-1.70-0.76-1.53-2.02-0.46-1.670.00-0.30E-0.55-1.69-0.480.00-1.18-1.03-1.15NaN-1.37-0.07NaN-1.85-2.36-1.19NaN-1.27-0.260.11 -0.52-0.96-0.70-0.88-1.42-1.36-0.15-0.56-2.42NaN-1.82-0.48-1.63-0.82NaN-1.63-1.30NaN-0.61-0.32-0.60NaN-0.42-1.90-1.90-0.30-1.03-2.05NaN-1.54NaN-1.78NaN-1.40-1.86-0.99-1.39-1.31-1.20-2.14-0.20-1.10H-1.23-1.54-2.21-1.85-1.52-2.41-2.15-0.24-1.35-0.07-1.22-0.49-2.17-0.47-0.17-1.77-1.47-0.57-0.34-0.78-0.42-0.62-1.57-1.21-0.74-1.64-1.57-1.37NaN-1.24-1.40-0.58-1.60-0.48-1.47-1.57-1.87NaN-0.71NaN-1.69-1.69-0.21-0.60NaN-0.51-0.39-0.33-0.71-0.64NaN0.00-1.55-0.58-1.64-1.15-2.12-0.90-0.57-1.47K0.00-1.57-1.55-0.27-1.79-1.60-1.69NaN-1.65-0.20-1.13-1.53-0.39-1.23NaN-1.160.00-0.79-0.34-0.83-0.27-0.74-0.36-1.080.00-1.63-0.40-1.84-1.52-1.89-0.46-0.60-1.33-3.02-2.09NaN-1.33-2.26-0.32NaN-1.44-1.28-1.49-1.52-1.74-0.58NaN-0.45-0.93-1.25NaN-0.79-1.33-1.40-2.17-1.37-1.51NaNNaN-1.46R-0.28-1.30-1.93-1.35-1.85-1.58-2.610.00-0.43-0.10-0.83-1.36-0.93-1.18-0.62-1.64-0.06-1.02-0.47-0.810.09-0.35-0.84-1.76-0.11 -1.45-1.04-1.04-0.39-1.61-1.41-1.50-0.45-1.66-1.62-1.92-2.16-1.47-2.04-1.73-1.43-1.29-1.11 -1.53-0.68-0.36-1.61-0.24-2.60-1.23-0.58-1.02-0.74-1.35-2.59-1.57-1.07-1.95-1.70-1.24AmiE I122L 241242243244245246247248249250251252253254255256257258259260261262263264265266267268269270271272273274275276277278279280281282283284285286287288289290291292293294295296297298299300Mutation RTLGECGEEEMGIQYAQLSLSQIRDARANDQSQNHLFKILHRGYSGLQASGDGDRGLAECSTOP *-1.40-1.38-1.39-1.24-0.52-0.50-1.71-0.44-0.64-0.53-1.00-1.39-1.58-0.63-0.67-1.40-0.55-0.63-1.42-1.93-0.58-0.40NaN-1.44-1.10-1.93-2.78-1.30-1.59-1.94-0.35-1.33-0.24-1.26-2.83-0.58-1.48-0.39-2.15-0.50-1.57-0.90-1.51-0.84-0.97-2.27-0.79-0.29-1.65-0.62-4.11 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-0.010.00-0.300.010.32-0.03-0.05-0.500.09-0.09-0.14S-0.39-0.72-1.00-0.84-0.72-0.64-0.65-0.75-1.43-0.20-1.08-0.21-1.07-0.58-0.64-0.73-0.15-0.46-0.58-1.13-0.27-1.43-0.20-0.640.00-0.01-0.04-0.040.060.070.06-0.26-0.20-0.05-0.060.14-0.03-0.08-0.03-0.05-0.04T-0.41-1.59-0.89-1.96-1.67-0.810.00-1.23-1.260.00-1.21-0.60-0.92-0.33-0.24-1.28-0.18-0.41-1.12-0.80-0.35-1.500.00-0.91-0.320.000.00-0.050.11 -0.03-0.01-0.38-0.20-0.02-0.100.14-0.09-0.310.21-0.06-0.08N-1.27-1.24-0.91-1.39-0.43-0.70-0.26-1.26-2.43-0.62-0.06-1.92-0.44-0.39-1.84-1.93-0.350.00-1.92-0.99-0.17-1.42-0.41-0.900.04-0.29-0.60-0.04-0.250.03-0.02-0.20-0.20-0.140.020.23-0.12-0.080.280.000.03Q-0.19-1.57-0.57-1.53-1.28-0.53-0.20NaNNaN0.09NaN-1.13-0.24-0.24-1.82-0.80-0.010.06-2.00-0.25-0.08-0.66-1.93-0.96-0.01-0.02-0.85-0.10-0.370.120.00-0.35-0.06-0.11 -0.040.200.03-0.130.19-0.07-0.04DNaN-1.520.03-1.57-1.21-1.58-0.84NaN-0.32-1.510.00-0.53-0.38-1.96-1.41-1.44-0.17-0.54-1.18-0.40-1.26-2.04NaN-2.28-0.32-0.35-0.72-0.08-0.340.020.11 -0.16-0.27-0.050.060.270.180.030.280.070.12E-1.44-1.270.00NaNNaN-1.27-0.82-1.35-0.91-0.70-0.16-0.890.00-0.26-0.53-1.400.00-0.35-0.490.00-0.49-1.20-1.01NaN-0.38-0.17-0.61-0.07-0.720.130.10-0.19-0.24-0.060.050.230.11 -0.230.270.00-0.01H-1.32-1.97-1.21-1.46-0.32-0.22-1.21-0.98-2.64-0.14-0.44-0.05-1.12-1.04-1.37-1.45-0.42-0.33-1.35-1.00-0.29-1.03NaN-0.35-0.06-0.09-1.00-0.10-0.44-0.05-0.27-0.48-0.27-0.08-0.130.08-0.03-0.240.19-0.15-0.11 K-1.41-1.35-0.41NaN-1.610.03-1.17-1.24-0.97-0.10-1.54-1.08-0.120.00-1.06-0.65-0.07-0.45-1.71-0.390.11 -1.54NaN-0.270.08-0.14-0.23-0.04-0.540.12-0.07-0.32-0.01-0.15-0.060.22-0.08-0.160.14-0.050.09R-0.87-1.57-1.72-1.74-1.610.00-1.38-0.28-1.87-0.29-1.35-1.59-1.13-0.13-1.530.00-0.07-0.33-2.09-0.500.00-1.34-2.030.000.04-0.23-0.60-0.26-0.650.01-0.33-0.46-0.18-0.43-0.140.00-0.43-0.360.07-0.17-0.10Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged AmiE I122L 123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960Mutation MRHGDISSSNDTVGVAVVNYKMPRLHTAAEVLDNARKIAEMIVGMKQGLPGMDLVVFPEYSTOP *-1.72-0.23-1.09-1.48-1.36-1.51NaN-0.20-0.16-1.44-1.61-2.35-1.34-1.10-1.59-1.47-1.78-1.43-1.39-0.52-0.64-1.86-1.34-1.52-1.11 -1.51-1.67-1.64-1.50-0.30-1.40-1.27-1.16-1.48-2.08-0.93-0.72-1.59NaN-0.57-1.67-1.23-1.48-2.47-1.55-0.51-0.71-1.70-1.42-1.73-1.74-1.56-1.62-1.11 -1.82-1.29-1.03NaN-0.25-0.10FNaNNaN-0.46NaN-1.64-0.46NaN-1.220.21-1.32-1.15-1.35NaN-1.35-1.35-1.79-1.59-1.39-1.87-0.29-1.80-1.48-1.12-1.40-0.25-0.88-0.93-1.23-0.60-1.27-0.72-0.68-0.68-1.42-1.66-0.80-0.60-0.50NaN-1.59-0.20-0.54-0.60-0.75-0.92-1.41-0.77-1.23-0.04-1.36-1.21-1.43-1.06-1.14-0.30-1.930.00-1.17-1.23-0.08WNaNNaNNaNNaN-1.77NaNNaN-0.21NaN-1.33-1.12NaN-1.09-0.85-1.64-0.95-1.14-1.44-1.15-0.61-1.89-3.09-1.39-3.41-1.70-0.81-1.82-1.22-0.64-1.70-1.08-1.65-0.54-1.80-1.31NaN-1.17-1.00-1.17-1.45-1.17-1.73-0.53-0.67-1.42-1.26-0.98-1.52-0.50-1.10-1.13-1.33-1.22-2.13NaNNaNNaNNaN-1.43NaNY-0.93NaN-0.23-1.87-0.57-1.45NaN-1.130.34-0.46-0.33-1.11 -1.76-2.08-1.83-1.41-1.50-2.18-0.460.00-1.49-1.23-1.71-1.08-1.20-0.18-0.78-0.75-0.52-1.18-1.28-0.72-0.32-1.11 -2.67-0.36-0.45-4.79NaN-1.06-0.93-1.36-0.34-0.74-1.46-1.76-0.59-0.94-0.01-1.12-1.47-1.60-0.54-1.55-1.79NaN-0.20NaN-1.520.00P-1.84-1.380.41-1.61-1.72-1.73NaN-0.57-0.360.03-1.65-0.93-1.46-1.22-1.28-1.19-1.49-1.54-1.89-1.35-0.10-0.980.00-1.23-1.32-0.69-1.040.10-0.35-1.28-1.43-0.32-1.21-2.09-0.74-0.56-1.34-1.38-0.38-1.73-1.34-1.69-0.87-1.14-1.47-1.39-1.00-1.67-0.390.00-1.56-1.36-1.59-0.65-1.22-1.43NaN0.00-1.54-2.35START M0.00NaNNaN-1.22-1.73-0.58NaN-1.440.06-0.27-2.54-0.66-1.00NaN-1.32-1.85-0.30-0.26-1.27-1.64-0.210.00-0.79-1.43-0.38NaN-0.82-0.35-0.23-0.49-1.430.03-0.29-1.35NaNNaN-0.41-0.97NaN-0.720.00-0.42-0.22-0.550.00-0.29-0.28-1.400.05-1.25-1.950.00-1.67-0.29-1.22-0.05NaN-0.85NaNNaNI-0.19-0.200.23-0.93-1.490.00-0.010.070.27-0.21-1.30-0.20-0.17-2.30-0.39-1.43-1.60-0.40-0.65-1.29-1.25-0.26-2.12-1.10-0.11 -1.14-0.72-0.74-0.35-1.63-0.260.09-0.40-0.78-1.60-0.85-0.430.00-1.06-1.65-0.150.000.03-1.85-0.31-1.67-0.59-1.42-0.13-1.04-1.50-0.16-1.60-0.78-0.19-0.03-0.15-1.47-1.12-0.40L-0.32-2.060.20-1.86-1.57-0.59NaN-0.36-0.14-0.26-1.64-1.34-0.45-1.33-0.61-1.29-1.12-0.75-1.46-1.45-1.13-0.69-0.37-1.010.00-0.56-0.92-0.68-0.43-1.17-1.140.00-0.33-1.53-1.26-0.30-0.51-0.64-1.50-1.15-0.25-0.40-0.30-0.79-0.09-1.87-0.51-1.440.00-0.53-1.830.24-1.420.00-0.59-0.45-0.06-0.28-1.44NaNV-0.29-1.270.16-0.29-0.59-0.13-1.22-0.36-0.38-0.35-0.58-1.530.00-0.330.00-0.130.000.00-1.59-1.45-0.66-0.61-0.98-0.75-0.17-1.31-1.03-0.12-0.25-0.380.00-0.16-0.16-1.69-0.15-0.59-0.48-0.25-0.10-0.62-0.250.090.00-0.65-0.45-1.71-0.39-1.02-0.52-0.95-0.66-0.37-0.56-0.840.000.00-0.23NaN-0.32NaNA-1.13NaN-0.270.08-0.78-1.45-0.660.050.180.17-1.25-0.16-0.33-0.54-0.540.00-0.56-0.61-1.55-1.22-0.28-1.73-0.97-0.78-0.52-1.05-0.390.000.00-1.01-0.62-0.43-0.05-1.180.00-0.20-0.15-1.620.00-0.13-1.26-1.12-0.33-0.10-1.08-0.89-0.10-0.39-1.13-0.75-1.23-1.07-1.82-1.82-0.26-0.31-1.24-0.42-1.22-1.22G-1.57-0.42-1.420.00-0.54-1.24-0.070.270.26-0.11 -0.52-0.60-0.750.00-0.83-0.96-0.85-0.48-1.32-1.67-0.59-2.43-1.91-0.93-1.33-1.23-0.28-0.09-0.04-0.30-0.81-0.72-0.15-1.08-0.60-0.29-0.56-1.64-0.06-0.21-1.12-1.92-0.420.00-1.80-2.05-0.600.00NaN-1.510.00-1.70-0.66-1.31-0.53-0.51-0.96-1.35-0.24NaNC-1.31-1.31-0.22-0.05-0.20-1.12-0.11 0.230.20-1.52-1.18-0.17-0.98-0.13-0.85-0.12-0.92-0.51-1.80-0.28-0.60-1.13-1.77-0.39-0.04-1.04-0.19-0.09-0.23-0.67-0.68-0.37-0.33-1.64-0.92-0.13-0.24-1.13NaN-0.62-0.63-0.82-0.15-0.17-1.19-1.36-0.44-0.55-0.62-1.01-0.55-0.40-1.88-1.97-0.63-0.54-0.33NaN-1.10-0.09S-1.35-0.430.040.06-1.48-0.990.000.000.00-0.22-1.42-0.20-1.47-0.38-1.41-0.48-1.55-1.41-0.74-1.71-0.26-1.42-0.22-0.41-0.50-0.530.040.120.04-0.56-1.40-0.68-0.15-0.46-0.43-0.08-0.21-0.88-0.15-0.14-1.30-0.91-0.45-0.14-1.54-1.06-0.20-1.10-1.28-0.27-0.90-2.26-1.44-1.65-1.28-1.23-0.20-0.22-1.71-0.84T-0.37-0.800.04-0.90-1.55-0.63-0.180.140.20-0.02-2.020.00-1.95-2.03-1.31-0.48-1.31-1.26-1.27-1.52-0.22-0.67-0.72-0.63-0.47-0.820.000.01-0.08-0.97-0.74-0.22-0.20-0.40-0.36-0.18-0.51-0.62-0.16-0.30-0.30-0.52-0.32-0.67-0.81-1.08-0.23-1.60-0.73-0.75-1.65-0.56-1.54-1.34-2.03-1.71-1.26-0.11 -1.37-2.06N-1.47-2.010.07-0.47-0.34-0.64-0.100.030.170.00-0.50-0.10-1.83-1.52-1.66-1.69-1.54-1.220.00-1.06-0.65-1.40-1.35-0.77-0.62-0.490.000.02-0.14-0.91-1.25-0.59-0.11 0.00-1.560.13-0.13-0.60NaN-0.01-1.90-0.60-0.52-0.12-1.37-0.55-0.29-1.53-1.62-1.39-1.75-2.42-0.53-1.36-2.13-1.00-1.57NaN-2.13-0.08Q-1.27-1.59-0.07-1.41-1.45-1.78NaN-1.120.120.19-1.51-0.62-2.37NaN-2.17-1.34-1.47-3.36-0.60-1.51-0.65-1.40-0.34-0.65-1.18-0.86-0.730.22-0.06-0.02-1.38-0.15-0.06-0.60-1.33-0.11 -0.22-1.77NaN-0.16-0.50-1.31-0.09-0.36-1.37-1.030.00-1.60-0.61-0.49-1.69-2.10-1.73-0.59-1.54NaNNaNNaN-0.29NaND-1.57-2.16-0.48-0.220.00-1.61NaN-0.850.41-0.060.00-1.90-1.11 -0.23-1.31-0.71-1.34-1.40-0.50-0.95-1.27-1.64-1.93-1.24-1.37-0.70-0.49-0.30-0.03-0.71-0.68-1.760.00-0.53-0.35-0.75-0.55-2.13-0.930.22NaN-3.10-0.42-0.43-1.93-2.17-0.65-0.82-1.50-1.37-0.28-1.320.00-2.06-0.17-1.90-0.96NaN-0.44-0.79E-1.38-1.76NaN-0.85-0.52-1.56NaN-1.13-0.21-0.12-0.46NaN-0.22-1.22-0.50-1.25-0.51-0.52-1.29-1.74-0.21-1.19-3.34-1.38-1.37NaN-0.82-0.52-0.090.00-1.44-0.800.00-0.73-1.85-0.21-0.23-1.35-0.080.00-1.39-1.82-0.10-0.71-1.50-0.37-0.35-1.88-2.08-1.17-2.37-1.44-0.22-1.46NaN-0.13NaN-0.850.00NaNHNaNNaN0.00-1.71-1.17-2.36NaN-1.550.26-0.26-0.85-1.16-1.55-1.17-1.80-1.52-2.18-2.21-1.11 -0.61-0.92-1.25-1.50-0.44-1.050.00-0.390.09-0.17-0.57-1.53-0.37-0.27-1.23-1.480.00-0.17-1.85-1.00-0.21-0.90-1.79-0.25-0.20-1.67-1.57-0.19-1.31-0.84-1.21-1.79-1.47-1.23-1.24-1.54-0.70-1.700.13NaN-0.46K-0.51-0.38-0.85NaN-1.44-1.34NaN0.14-1.000.03-1.07-1.47-1.26-1.45-1.64-1.72-1.47-1.42-0.50-1.750.00-0.90-1.31-0.36-1.45-0.46-0.310.43-0.07-0.32-1.730.02-0.30-0.58-1.980.120.00-1.52NaN-0.21-0.24-1.88-0.50-0.63-0.740.000.07-1.51-1.14-1.23-2.41-0.65-2.17-2.79NaNNaN-0.85NaN-0.17-1.43R-0.750.00-0.21-0.84-1.29-1.280.00-0.40-1.450.02-1.38-1.49-1.64-0.54-1.36-1.55-1.51-1.28-1.36-1.33-0.17-1.38-0.940.00-1.46-0.24-0.340.36-0.13-1.06-1.240.04-0.29-1.54-1.620.00-0.07-1.43-1.76-0.20-0.62-1.44-0.58-0.37-1.35-0.570.03-1.39-0.80-0.99-1.26-0.35-1.67-1.25-1.26-3.39-1.06-0.46-1.16-1.40AmiE I122L 616263646566676869707172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110 111112 113 114 115 116 117 118 119 120Mutation SLQGIMYDPAEMMETAVAIPGEETEIFSRACRKANVWGVFSLTGERHEEHPRKAPYNTLVSTOP *-1.17-1.03-0.49NaN-1.98-1.57-0.48-1.72-1.56-1.43-0.35-1.46-1.37-0.66-1.30-1.82-1.26-1.63-1.44-1.85-1.60-0.76-0.57-1.30-0.13-1.67-1.60-1.35-1.92-1.68-0.27-0.77-0.71-1.72-2.05-1.76-0.54-2.32-1.75-1.81-1.83-1.95-1.87-2.23-0.83-1.31-1.62-0.63-0.28-1.35-1.53-1.00-0.61-2.33NaNNaNNaNNaNNaNNaNF-0.18-0.36-1.55NaN-0.44-1.35-0.17-1.30-0.49-0.40-0.49-1.02-0.26-1.31-0.59-1.32-0.96-0.57-0.76-1.53-1.98-0.68-1.38-1.97-0.73-0.620.00-1.00-0.69-1.73-0.31-1.60-1.27-1.63-3.66-0.79-0.93-1.78-1.470.00-0.33-0.25-2.26-1.59-1.64-1.12-1.58-1.82-0.66-1.26-1.68-0.18-2.02-0.90NaNNaNNaNNaNNaNNaNWNaN-1.00-2.67-0.85-1.33NaN-0.66-1.30-0.38-0.44-0.76-1.24-0.72-1.56-1.22-1.32NaN-0.51-1.64-1.22-1.45-0.99-1.51-1.37-1.54-0.84-0.37NaN-0.06NaN-0.17-1.50-1.45-2.74NaN-1.200.00-1.50NaN-1.17NaN-1.81-3.53-1.80-2.07NaNNaN-1.71NaN-1.24NaN-0.59NaNNaNNaNNaNNaNNaNNaNNaNY-0.11 -1.70-2.15NaN-1.47-1.790.00-0.51-0.27-0.25-0.84-1.19-0.08-0.76-0.64-2.61NaN-0.25-1.38-1.20-1.57-0.50-1.57-1.49-1.00-1.52-0.31NaN-0.26-1.31-0.23-1.29-0.78-1.76-0.42-2.66-0.92-2.15NaN-0.51-0.77-1.30-1.73-1.75-1.59NaN-0.41-2.98-0.70-0.42-2.44-0.01-1.49-0.74NaNNaNNaNNaNNaNNaNP-0.26-1.55-1.09-1.53-0.97-0.43-1.05-1.440.00-0.82-1.09-1.73-1.87-1.27-1.15-1.00-1.24-0.17-1.530.00-1.620.34-1.33-1.38-1.54-1.95-1.91-1.33-0.89-1.01-1.35-1.62-1.18-1.50-1.89-2.15-1.91-2.11 -2.30-1.88-0.75-0.53-1.57-1.97-2.33-0.56-1.47-0.61-1.84-1.410.00-2.16-1.52-0.49NaNNaNNaNNaNNaNNaNSTART MNaN-0.76-1.45NaN-0.860.00-0.73-1.11 -0.33-0.12-0.230.000.00-0.39-0.30NaN-0.04-0.04-0.64-1.34-2.10-0.21-1.46-0.25-0.50-0.63NaN-1.000.21-1.17NaN-1.00-0.21-1.15-2.56NaN-1.45NaN-0.22NaN-1.58-0.18-1.66-3.28-4.70NaN-1.77-1.23-0.50-1.84-1.73-0.16-0.35-0.05NaNNaNNaNNaNNaNNaNI-1.03-0.60-1.42-1.820.00-0.17-0.69-1.12-0.75-0.05-0.48-0.28-0.09-0.85-0.60-1.72-0.64-0.400.00-1.50-1.44-0.04-1.490.17-1.060.00-0.27-0.36-0.54NaN-1.30-0.59-0.41-2.53-0.92-0.59-1.76-1.56-0.40-0.46-2.37-0.07-0.80-1.82-2.27-0.67-3.22-1.18-0.96-1.74-3.31-0.22-1.76-1.73NaNNaNNaNNaNNaNNaNL-1.090.00-0.88-1.33-0.56-0.52-0.49-1.18-0.51-0.10-0.27-0.43-0.03-0.64-0.23-1.45-0.33-0.20-0.67-0.44-1.55-0.15-1.43-0.29-0.68-0.77-0.05-1.28-0.26-2.47-1.77-1.07-0.33-1.64-1.86-0.94-0.51-1.74-0.54-0.39-2.310.00-2.03-1.66-1.64-0.44-0.73-1.57-0.42-0.77-0.80-0.09-2.22-0.80NaNNaNNaNNaNNaNNaNV-1.57-0.87-1.62-0.23-0.27-0.13-0.76-0.58-0.470.00-0.26-0.17-0.19-0.47-0.47-0.140.00-0.080.10-1.50-0.770.06-0.970.15-0.26-0.32-0.41-1.47-0.33-0.32-1.43-0.76-0.48-0.79-1.540.00-3.05-0.510.00-0.92-1.89-0.24-1.67-0.68-0.78-1.32-1.82-0.71-0.29-1.78-2.05-0.21-2.15-0.19NaNNaNNaNNaNNaNNaNA-0.34-1.73-1.55-0.83-0.67-0.45-0.91-0.77-0.170.00-1.21-0.64-0.980.05-0.410.00-0.200.00-0.34-0.83-1.250.12-0.45-0.74-0.02-1.47-1.490.350.310.00-1.58-0.430.030.00-1.12-0.70-1.69-0.84-0.51-2.46-1.00-0.94-0.64-1.14-1.58-0.73-2.65-0.88-0.48-1.98-1.10-0.09-1.360.00NaNNaNNaNNaNNaNNaNG-1.00-1.76-1.160.00-1.48-1.42-2.03-0.57-0.380.01-0.33-1.86-1.20-0.17-0.84-0.68-0.65-0.31-1.48-0.890.00-0.48-0.72-0.88-0.20-1.96NaN0.01-0.27-0.67-0.53-0.61-0.92-1.08-0.08-0.53-0.870.00-0.77-1.49-2.30-1.34-1.670.00-0.85-0.42-1.71-0.58-0.24-4.22-2.46-0.47-1.71-0.71NaNNaNNaNNaNNaNNaNC-0.27-0.94-1.88-0.17-0.51-0.70-0.08-0.59-0.46-0.13-0.45-0.27-0.96-0.38-0.19-0.44-0.400.080.15-1.45-0.21-0.28-1.30-0.59-0.63-1.06-0.22-0.20-0.06-1.270.00-0.81-0.52-0.88-0.830.02-0.45-0.43-1.34-0.51-1.11 -0.41-1.99-0.63-2.11 -0.15-3.50-1.35-0.26-2.15-1.40-0.31-1.22-0.51NaNNaNNaNNaNNaNNaNS0.00-0.48-0.75-0.54-0.77-0.48-0.56-0.41-0.17-0.01-0.44-1.20-1.06-0.01-0.48-0.35-1.430.16-0.94-0.53-0.70-0.26-1.25-0.77-0.54-1.51-0.270.000.06-0.40-0.60-0.55-0.17-0.77-0.50-1.26-1.30-0.84-1.61-0.590.00-1.67-0.92-0.54-2.01-0.28-1.74-1.23-0.65-2.00-0.83-0.18-1.51-0.10NaNNaNNaNNaNNaNNaNT-0.340.09-0.77-1.56-0.76-0.34-0.51-0.66-0.120.02-0.37-0.62-0.45-0.170.00-0.64-1.350.05-0.79-0.55-1.59-0.21-1.430.00-0.68-0.61-1.60-0.61-0.06-0.42-1.49-1.01-0.04-0.66-0.66-0.30-2.27-1.66-1.80-1.78-0.93-1.440.00-1.82-2.01-1.45-2.27-1.64-0.91-1.71-0.78-0.15-0.94-0.10NaNNaNNaNNaNNaNNaNN-1.06-1.09-1.76NaN-0.59-0.51-0.26-0.11 -0.120.06-0.38-1.58-0.970.06-0.51-1.67-1.470.00-0.90-1.31-1.83-0.30-1.38-1.78-0.45-0.81NaN-0.300.16-1.78-2.00-0.83-0.25-1.210.00-1.47-1.36-1.49-1.44-1.61-1.86-2.49-0.98-2.37-2.05NaN-0.38-1.40-0.39-0.54-2.740.05-0.60-0.03NaNNaNNaNNaNNaNNaNQ-1.08-1.250.00-1.73-1.12-0.19-0.87-0.730.040.12-0.12-0.59-0.970.05-1.07-1.62-0.900.04-1.45-0.13-1.690.08-0.61-1.29-0.16-1.07NaN-1.190.28-2.05-2.22-0.180.35NaN-0.40-5.01-1.26-3.41-1.74-1.84-1.96-0.57-1.75-2.00-1.04-0.13-0.49-0.69-0.18-0.33-1.63-0.10-1.26-0.22NaNNaNNaNNaNNaNNaND-0.59-1.18-1.39-0.30-1.45-1.19-0.560.00-0.61-0.04-0.22-1.59-1.730.03-1.31-0.35-1.73-0.45-1.30-0.95-0.67-0.22-0.15-1.53-0.40-1.64-0.71NaN0.30-0.36NaN-1.280.21-1.76-0.36-0.61-1.90-0.27-1.74-1.38-1.88NaN-2.15-0.20-0.93NaN-0.89-0.48-0.01-1.04-1.53-0.32-1.67-1.28NaNNaNNaNNaNNaNNaNENaN-1.02-0.93-1.13-1.61-1.60-0.76-0.23-0.380.010.00-1.82-1.040.00-1.43-1.64-0.160.05-1.40-1.48-1.520.000.00-1.510.00-1.28-1.27NaN0.36-1.33-1.00-0.850.27-0.67-1.06-1.63-1.69-1.57-0.45-1.73-3.91-1.41-2.35-3.960.00NaN-1.700.000.00-1.67NaN-0.32-0.24-0.24NaNNaNNaNNaNNaNNaNHNaN-1.03-0.21-1.57-1.39-0.58-0.10-0.600.040.04-0.42-1.28-0.91-0.16-0.52-1.35-1.90-0.13-1.40-1.19-1.76-0.26-0.56-1.51-0.50-1.30-1.22-2.45-0.01-1.54-1.86-1.12-0.40-2.12-0.64-1.61-1.44-2.09-1.84-1.58-1.61-2.04-1.81-1.74-1.71-0.080.00-1.37-0.270.00-0.490.09-2.07-0.42NaNNaNNaNNaNNaNNaNKNaN-1.71-0.98-1.43-1.43-0.25-1.10-0.550.040.20-0.25-0.56-0.30-0.04-0.37-1.53-1.120.00-1.41-1.41-1.900.09-0.75-0.76-0.15-1.76-1.00-1.000.08-2.29-1.450.080.00-1.47-0.27-1.41-1.34-2.28-2.53NaN-2.59-2.63-1.96-1.74-0.63-0.28-2.03-0.56-0.32-1.62-2.030.060.00-0.48NaNNaNNaNNaNNaNNaNR-1.39-1.37-0.84-0.97-1.46-0.53-0.60-0.860.050.07-0.37-0.21-0.440.00-0.31-1.64-1.86-0.14-1.50-0.77-1.250.09-1.47-1.17-0.33-1.80-1.67-0.230.00-1.67-0.420.00-0.09-2.09-0.73-2.09-0.69-1.41-5.74-2.58-1.72-0.96-1.88-1.45-2.500.00-0.67-1.35-0.95-0.71-1.460.00-0.50-0.08NaNNaNNaNNaNNaNNaNAmiE I122L 121122123124125126127128129130131132133134135136137138139140141142143144145146147148149150151152153154155156157158159160161162163164165166167168169170171172173174175176177178179180Mutation LLDNNGEIVQKYRKIIPWCPIEGWYPGGQTYVSEGPKGMKISLIICDDGNYPEIWRDCAMSTOP *NaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.96NaN-0.37-0.70-2.37-1.71-1.61-0.44-0.58NaN-2.57-0.94-1.65-0.13-0.72-3.50-2.00-2.19-0.21-1.94-1.01-1.58-1.82-0.48-1.96NaN-0.46NaNNaN-0.61-1.79-0.70-0.64-1.93-2.01-0.60-1.34-1.93-1.77-1.78-0.36-1.93-0.34-1.40-0.17-0.38-1.25-0.13-2.68-1.36FNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-2.08NaN-1.21-2.25-0.340.09-1.91-0.68-0.63NaN-0.30-2.03-0.13-0.77-0.45-1.52-1.95-1.30-0.94-1.520.06-1.33-0.68-0.93-2.00NaNNaNNaNNaN-1.00-0.62-1.84-0.35-0.30-0.35-0.64-1.68-2.22NaN-1.12-0.25-2.39NaN-0.35-1.76-1.88-1.90-0.39-1.43-1.13WNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-2.64NaNNaNNaN-1.23-1.49-1.230.00-0.41NaN-1.29NaN-0.560.00-2.22-1.39-1.60NaNNaN-3.03-0.84-1.78-1.65-1.22-1.49NaNNaNNaNNaN-1.58NaN-1.32NaN-1.31-1.39-0.55-1.50NaN-2.29-1.06-0.74-2.07-1.15NaN0.00-1.11 NaN-0.17NaN-0.68YNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-2.37NaN-2.69-1.80-1.70-0.53-2.11 -0.51-0.50NaN-0.85-1.49-0.28NaN0.00-1.70-2.58-1.88-0.82-2.160.00-1.72-0.67-0.57-3.02NaN-2.65-1.17-0.74-1.11 -1.75-1.45-3.96-2.56-1.91-0.57-0.37-0.34-2.39-0.580.00-1.36-0.72-2.02NaN-1.82-0.24-0.06NaN-0.85PNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-2.01NaN-1.70-2.07-2.00-1.730.00-2.24-1.820.00-1.57-1.490.03-1.31-1.850.00-1.69-1.82-1.27-1.48-2.13-2.02-0.89-0.24-1.850.00-2.41-2.22-1.59-1.73-1.79-0.78-1.53-1.46-1.77-1.51-1.60-1.74-1.80-1.67-1.340.00-1.61-1.39-0.99-1.54-1.35-1.45-0.68-1.87START MNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.61NaNNaN-0.45-0.170.18-1.40-1.63-1.63NaN-0.58NaN-0.10NaN-1.48-1.35NaN-0.15-0.25NaN-1.20-1.93-2.05-0.42-1.24NaN-0.86NaN0.00-0.28-0.24NaN-0.07-1.16-0.40-1.60-2.19NaN-1.88-1.54-0.95-1.10-1.43-0.18-1.49-1.59NaNNaNNaN0.00INaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.40NaN-0.63-3.230.000.00-2.14-2.07-1.70NaN0.00-2.57-0.32NaN-1.95-1.42-1.92-0.77-1.20-0.66-1.76-0.69-1.86-0.08-1.72NaN-0.48-2.22-0.07-1.180.00-1.45-0.620.000.00-2.34-2.05-1.63-1.60-0.21-1.68-1.29-1.620.00-1.06-0.61-1.58-1.54-1.97-0.27LNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.33NaN-2.16-1.85-0.11 0.16-0.81-1.01-1.78-0.16-0.46-1.77-0.25-0.19-2.14-0.50-1.63-0.55-0.36-1.89-1.51-1.40-1.94-0.25-2.00-0.18-1.77-2.24-0.07-0.66-0.41-0.780.00-0.99-0.41-1.99-3.07-2.58-1.94-1.83-1.48-0.42-1.68-0.84-0.19-1.64-2.71-1.50-1.33-0.41VNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.26NaN-2.11 -2.23-0.540.05-1.96-1.73-0.68-1.760.09-0.71-0.17NaN-2.05-1.75-0.82-0.19-0.72-1.17-1.860.00-2.530.02-1.24NaN-1.88-0.30-0.18-1.25-0.17-1.94-0.96-0.300.10-1.94-0.52-0.20-0.92NaN-1.90-1.65-0.260.02-1.02-1.51-0.16-1.49-0.05-0.29ANaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.74NaN-1.83-2.08-2.34-0.41-1.19-1.31-0.57-0.40-0.72-1.80-0.04NaN-2.76-0.96-1.380.02-0.16-1.00-2.27-0.71-0.90-0.20-1.68-0.49-1.49-0.60-1.31-1.04-1.86-0.68-1.52-2.24-0.70-1.66-1.16-1.40-1.41-1.70-1.58-0.91-0.14-1.68-1.80-2.03-0.56-1.680.00-0.82GNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.72NaN-0.36-3.19-1.74-1.55-2.15-1.42-0.61-1.15-1.39-0.570.00NaN-1.64-1.700.000.00-0.43-3.04-1.95-1.07-1.60-0.410.00-1.73-2.450.00NaN-1.61-1.70-1.86-1.47-1.37-2.80-1.12-0.51-0.300.00-2.54-1.35-1.43-0.24-1.30-0.41-0.22-0.12-0.26-0.32-1.21CNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.43NaNNaN-1.47-2.460.04-3.31-0.370.00NaN-0.97-9.04-0.07-0.51-0.49-2.63-0.47-0.11 -0.07-2.93-0.42-0.27-0.59-0.29-0.77-1.73-2.05-0.20NaN-1.02-1.15-1.68-0.41-1.190.390.00-1.18NaN-0.48-1.20-0.18-1.25NaN-0.87-0.07-1.69-1.330.00-1.13-1.65SNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.81NaN-0.88-1.73-1.53-0.39-0.86-1.41-0.54-0.23-0.69-2.150.06-0.29-1.00-0.60-0.940.10-0.09-0.92-1.48-1.840.00-0.18-1.33-0.35-2.05-0.31-1.59-1.460.050.00-0.65-0.86-0.74-0.80-1.41-1.76-0.54-0.520.40-0.47-1.44-0.91-0.53-0.41-1.89-0.24-0.47-1.19TNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.07NaN-0.95-1.50-0.77-0.87-0.69-1.610.01-0.35-0.40-2.22-0.04NaN-0.56-0.73-1.850.14-0.100.00-1.45-1.40-0.050.05-1.61-0.53-2.79-1.24-0.22-1.20-0.65-0.93-1.43-0.73-0.18-2.15-1.47-2.25-2.19-1.04-1.42-0.25-2.39-0.25-2.29-1.26NaN-1.27-0.26-0.45NNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.97NaN-2.42-0.63-0.62-0.14-1.71-3.33-1.18NaN-0.54-2.630.03NaN-0.58-1.59-1.220.25-0.03-2.22-0.96-2.26-0.71-0.04-1.91NaN-0.31NaN-1.09-0.76-0.81-2.29-1.66-0.41-0.45-2.67-0.31-0.50-1.680.00-0.72-1.37-1.06-0.54NaN-1.34-0.44NaN-1.88-1.83QNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.89NaN-1.51-1.16-1.39-1.46-0.54-1.73-2.02-0.21-0.64-0.960.14NaN-2.96-0.44-2.080.010.00-1.90-1.95-1.76-2.57-0.16-1.99-0.23NaNNaNNaN-0.72-1.33-1.93-1.77NaN-1.50-1.73-1.70NaN-1.91-2.07-2.76-0.31-0.60-0.97-1.24-1.58-2.62-0.81-1.57-1.87DNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-3.46NaN-1.33-2.40-2.65-1.94-1.99-2.88-0.58NaN-2.80-0.43-0.45NaN-0.97-1.97-0.680.00-0.55-2.24-1.22-0.80-1.090.05-0.71NaN-2.05-0.12NaN-1.78-1.59-1.47-3.00-1.31-1.88-2.270.000.00-0.69-0.24-0.77-1.70-0.37-1.05-1.07-1.830.00-0.76-0.21-1.54ENaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.60NaN-1.78-0.32-1.27-1.94-1.96-2.08-1.51NaN-3.190.00-0.45NaN-4.06-2.31-1.950.07-0.10-2.25-1.47-1.71-7.100.00-2.47-1.31-0.41NaNNaN-0.44-1.60-1.75-1.85NaN-1.57NaN-1.87-0.40-1.68-0.87-2.23-1.030.00-1.42NaN-1.12-0.20NaN-2.67-1.85HNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.82NaN-1.03-1.71-1.92-0.22-1.80-1.52-1.62-1.95-0.52-2.460.01NaN-0.48-1.93-1.62-0.03-0.16-1.68-0.48-2.13-2.12-0.29-1.83-2.17-2.11 -1.60-0.75-1.21-1.49-2.14-2.07-1.29-1.30-1.83-0.85-0.68-2.21-1.49-0.29-1.46-1.19NaN-1.49-2.00-0.53-1.19NaN-2.95KNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN0.00NaN-0.550.00-3.05-2.39-1.53-2.04-3.58-1.22-0.67-0.64-0.18NaN-1.71-2.07-1.400.11 -0.16-1.03-2.54-1.64-1.71-0.22-1.62-1.810.00-1.31-0.190.00-2.26-1.91-1.90-2.02-1.67-1.67NaN-1.57-2.01-0.13-1.15-1.12-0.23-2.62-1.78-0.72NaN-1.22-1.50-0.53RNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.15NaN0.00-0.70-1.82-1.72-1.30-0.83-0.77-0.36-1.08-2.02-0.13-0.10-2.16-0.84-1.92-0.09-0.36-1.32-1.88-1.70-1.79-0.27-1.72-0.77-0.53-0.63-0.68-0.26-1.82-1.71-1.70-2.60-2.29-0.72-2.09-3.85-1.38-2.39-1.45-1.03-1.70-1.78-0.270.00-1.70-0.18-1.69-0.54AmiE I122L 181182183184185186187188189190191192193194195196197198199200201202203204205206207208209210211 212213214215216217218219220221222223224225226227228229230231232233234235236237238239240Mutation KGAELIVRCQGYMYPAKDQQVMMAKAMAWANNCYVAVANAAGFDGVYSYFGHSAIIGFDGSTOP *-0.59-1.59-1.76-0.29-1.05-1.34-1.25-2.24-0.52-0.08-1.00-0.54-1.95-0.25NaN-2.82-0.39-1.71-0.35-0.77-2.48-1.85-1.57-3.25-0.56-2.26-1.49-1.74-0.15-1.41-2.77-1.46-0.35-0.48-1.80-1.21-1.59NaNNaN-0.84-2.20-1.99-1.58-1.32-1.54-2.14-1.00-1.42-0.30-2.11 NaN-1.86-1.54-1.57-1.55-1.57-1.80-1.77NaN-1.40F-1.27-1.44-1.57NaN-0.50-0.26-0.55-1.15-0.34-1.11 -1.76-0.08-2.19-0.39-1.63-1.34-1.55-0.68-1.37-1.52-0.46-0.90-1.27-2.31-1.46-1.84-1.51NaN-0.46-1.52-1.81-1.21-0.31-0.43-0.59-1.70-1.59-1.46-1.49NaN-1.65-2.440.00-1.49-1.43-0.21-0.03NaN-0.040.00NaN-1.25-2.21-1.33-0.68-0.26-1.270.00-1.15-1.39W-1.45-2.57-1.25NaN-0.79-1.41-2.05NaN-0.44NaN-1.38-1.56-1.69-1.38-1.29NaN-0.62-1.16NaN-1.99-0.70-0.56-1.24NaNNaN-0.96-1.98NaN0.00-2.02NaN-1.46-0.68NaN-1.30NaN-1.61-1.15-0.98NaNNaN-1.42-0.28-0.84NaNNaNNaNNaNNaN-0.34NaNNaN-1.02-1.35-2.00-1.83-1.49NaNNaN-1.00Y-1.73-1.67-1.47-1.30-1.97-1.46-2.18-1.65-0.10NaN-1.320.00-1.050.00-0.98-1.70NaN-0.38-1.23-2.30-0.63-0.87-1.46-2.54-1.54-2.94-1.30NaN-0.54-1.47-0.41-0.61-0.350.00-1.50-1.76-2.17-2.38-0.35NaN-1.00-1.74-0.18-0.25NaN-0.960.00-0.910.00-0.03NaN-0.17-1.58-1.88-1.650.21-2.26-0.39-0.24-1.90P-1.51-1.66-1.31-2.34-1.80-1.61-1.50-0.92-0.92-0.46-1.82-1.37-0.08-1.600.00-0.89-1.87-0.88-0.41-1.06-1.61-1.40-1.60-0.75-1.10-0.79-2.06-0.59-1.76-1.16-1.83-1.38-1.49-1.72-1.67-0.93-1.79-0.76-1.15-0.54-0.79-1.51-1.61-1.47-1.71-1.20-2.19-1.51-1.16-1.23-1.12-0.88-1.61-1.17-1.83-1.61-1.59-1.60-1.48-1.70START M-0.44-1.59-2.00NaN-0.69-0.44-1.83NaN-1.51NaN-0.93-1.570.00-1.15NaN-1.17-0.40-0.76-0.58-1.280.310.000.00-1.41-0.01-1.190.00-0.98-1.30-1.97-1.94-0.37-1.06-1.40-1.17NaN-1.50NaN-1.13NaNNaN-1.20-1.23NaNNaN-0.66-0.74-1.07NaN-0.74NaN-0.95-2.260.19-6.33-0.80-2.21-1.03NaN-1.86I-1.63-1.53-3.68-1.76-0.950.00-0.06NaN-1.34-1.54-2.02-2.10-0.35-1.82-1.08-1.72-0.45-0.98-0.36-1.500.20-0.07-0.26-1.53-1.35-0.88-0.19-2.02-1.11 -1.20-0.45-0.37-0.86-1.25-0.40-1.56-0.33-1.58-0.39-0.81-1.44-1.56-1.19-1.97NaN-0.10-1.82-0.33-1.61-0.52NaN-1.28-0.67-0.550.000.00-1.80-0.94-1.28-2.27L-1.47-1.72-1.78-1.610.00-0.90-0.94-0.47-1.75-0.18-1.76-1.80-0.66-1.31-0.36-1.34-0.91-0.96-0.54-1.180.17-0.08-0.30-1.75-1.76-1.52-0.66NaN-0.37-1.60-1.77-0.70-1.25-1.71-0.96-1.36-0.56-3.12-1.66-1.51-1.74-1.30-0.66-1.70-2.51-0.49-1.17-1.83-2.39-0.53-1.94-0.52-2.51-0.42-0.57-1.12-1.96-0.51-1.37-1.57V-1.68-0.83-0.19-0.21-0.93-0.270.00-1.46-1.29-1.11 -0.23-1.75-0.42-2.99-1.26-0.15-0.87-0.49-0.34-1.460.00-0.19-0.25-0.29-1.35-0.08-0.44-0.12-1.66-0.32-1.63-0.370.03-1.780.00-0.400.00-0.17-1.63-0.15-0.23-1.02-1.10-0.24-0.210.00-1.56NaN-0.86-0.78-0.63-3.13-1.43-0.34-0.53-0.52-0.57-0.53-0.09-0.44A-1.33-1.310.00-1.21-1.46-2.10-0.46-1.24-0.080.07-0.51-1.39-0.71-0.16-0.580.00-1.03-0.71-0.18-1.00-0.07-0.09-1.290.00-2.380.00-1.930.00-1.900.00-1.70-0.95-0.52-1.59-0.570.00-0.540.00-1.260.000.00-1.58-1.31-1.13-0.74-0.26-0.87-0.25-1.09-1.66-0.41-1.23-1.510.00-1.66-1.82-0.59-2.04-0.61-0.94G-1.000.00-1.10-0.27-1.53-1.70-0.70-0.41-0.81-0.960.00-1.35-0.47-1.93-2.07-0.34-1.60-0.63-1.43-1.46-0.16-0.38-1.09-0.61-1.86-0.61-1.51-0.36-0.83-1.05-1.47-0.62-0.50-1.82-0.86-0.83-0.98-0.69-2.78-0.24-0.640.00-1.52-0.540.000.02-1.32-0.16-1.78-2.070.00-0.65-0.97-0.65-1.51-1.530.00-1.54-0.160.00C-1.39-0.59-1.15-1.84-1.09-2.05-0.39-0.200.00-0.22-0.21-0.39-0.46-0.28-1.540.04-0.81-1.00-0.81-1.550.07-0.15-2.230.10-0.930.13-1.18-1.74-0.11 -0.32-3.78-0.330.00-0.32-1.46-0.81-0.17NaNNaNNaN-0.84-0.62-0.53-0.86-0.18-0.20-0.04-0.12-0.02-0.31-0.25-0.76-0.440.14-1.48-1.44-0.43-0.22-1.04-0.26S-1.21-1.14-0.64-1.68-0.78-0.82-1.64-0.500.01-0.06-0.37-1.32-0.78-0.16-0.37-0.18-0.57-0.58-0.77-1.180.02-0.40-1.970.16-1.14-0.08-2.07-0.35-0.71-0.83-0.69-0.31-0.35-1.60-1.93-0.61-1.03-0.29-0.48-0.200.00-1.24-0.62-1.45-0.22-0.65-0.840.00-1.34-0.15-0.05-1.430.00-0.04-3.08-0.99-0.19-0.52-1.84-0.44T-1.10-1.56-0.50-1.68-2.17-0.54-1.95-1.77-1.74-0.37-1.08-1.52-0.46-0.99-0.24-0.30-0.97-0.68-0.45-1.410.13-0.03-0.50-0.08-0.73-0.01-0.49-0.22-1.68-0.53-1.28-0.24-0.15-1.45-1.49-0.23-0.39-0.21-0.55-0.190.10-1.88-0.95-1.46-1.33-0.09-1.73-0.29-0.92-0.41-1.72-1.20-1.27-0.08-0.99-0.99-1.38-1.36-1.61-1.54N-0.56-1.55-2.63-1.19-1.82-0.35-1.79-1.45-1.93-0.66-1.90-0.80-0.85-0.09-1.49-0.48-0.47-0.40-1.29-0.76-0.07-0.01NaN-0.16-0.18-1.20-1.06-1.51-2.09-2.140.000.00-1.55-1.04-1.59-1.57-1.72-1.480.00NaN-1.89-1.59-0.47-0.35-1.82-0.97-0.53-0.28-0.26-1.92-0.84-0.17-0.68-1.21-0.78-0.75-1.03-1.77-0.23-1.27Q-0.67-1.49NaN-0.45-1.90-1.64-1.75-1.42-1.690.00-1.19-1.63-1.15-0.04NaN-0.86-0.48-0.550.000.000.09-0.29-1.12-0.76-0.74-1.56-1.43NaN-1.70-1.63-1.430.12NaN-1.73-1.82-1.75-0.34-1.56-1.30NaN-1.90-3.15-1.06-2.17-0.97-0.66-1.84-1.30-1.35NaNNaN-0.50-1.570.14-1.75-1.81-1.45-1.15NaN-1.60D-1.66-0.64-1.32-0.26-5.05-1.43-0.65-1.47-2.08-1.19-0.14-0.84-2.57-0.92-1.39-0.43-1.190.00-1.87-1.44-0.49-1.40-1.82-0.45-1.56-2.29-2.33-0.18-1.28-1.50-0.40-0.59-0.98-4.36-0.42-0.51-1.75-2.02-0.23-0.16-1.23-0.87-1.910.00-0.22-0.31-0.62-1.16-0.29-1.14-0.05-0.54-1.70-0.76-1.53-2.02-0.46-1.670.00-0.30E-0.55-1.69-0.480.00-1.18-1.03-1.15NaN-1.37-0.07NaN-1.85-2.36-1.19NaN-1.27-0.260.11 -0.52-0.96-0.70-0.88-1.42-1.36-0.15-0.56-2.42NaN-1.82-0.48-1.63-0.82NaN-1.63-1.30NaN-0.61-0.32-0.60NaN-0.42-1.90-1.90-0.30-1.03-2.05NaN-1.54NaN-1.78NaN-1.40-1.86-0.99-1.39-1.31-1.20-2.14-0.20-1.10H-1.23-1.54-2.21-1.85-1.52-2.41-2.15-0.24-1.35-0.07-1.22-0.49-2.17-0.47-0.17-1.77-1.47-0.57-0.34-0.78-0.42-0.62-1.57-1.21-0.74-1.64-1.57-1.37NaN-1.24-1.40-0.58-1.60-0.48-1.47-1.57-1.87NaN-0.71NaN-1.69-1.69-0.21-0.60NaN-0.51-0.39-0.33-0.71-0.64NaN0.00-1.55-0.58-1.64-1.15-2.12-0.90-0.57-1.47K0.00-1.57-1.55-0.27-1.79-1.60-1.69NaN-1.65-0.20-1.13-1.53-0.39-1.23NaN-1.160.00-0.79-0.34-0.83-0.27-0.74-0.36-1.080.00-1.63-0.40-1.84-1.52-1.89-0.46-0.60-1.33-3.02-2.09NaN-1.33-2.26-0.32NaN-1.44-1.28-1.49-1.52-1.74-0.58NaN-0.45-0.93-1.25NaN-0.79-1.33-1.40-2.17-1.37-1.51NaNNaN-1.46R-0.28-1.30-1.93-1.35-1.85-1.58-2.610.00-0.43-0.10-0.83-1.36-0.93-1.18-0.62-1.64-0.06-1.02-0.47-0.810.09-0.35-0.84-1.76-0.11 -1.45-1.04-1.04-0.39-1.61-1.41-1.50-0.45-1.66-1.62-1.92-2.16-1.47-2.04-1.73-1.43-1.29-1.11 -1.53-0.68-0.36-1.61-0.24-2.60-1.23-0.58-1.02-0.74-1.35-2.59-1.57-1.07-1.95-1.70-1.24AmiE I122L 241242243244245246247248249250251252253254255256257258259260261262263264265266267268269270271272273274275276277278279280281282283284285286287288289290291292293294295296297298299300Mutation RTLGECGEEEMGIQYAQLSLSQIRDARANDQSQNHLFKILHRGYSGLQASGDGDRGLAECSTOP *-1.40-1.38-1.39-1.24-0.52-0.50-1.71-0.44-0.64-0.53-1.00-1.39-1.58-0.63-0.67-1.40-0.55-0.63-1.42-1.93-0.58-0.40NaN-1.44-1.10-1.93-2.78-1.30-1.59-1.94-0.35-1.33-0.24-1.26-2.83-0.58-1.48-0.39-2.15-0.50-1.57-0.90-1.51-0.84-0.97-2.27-0.79-0.29-1.65-0.62-4.11 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-0.010.00-0.300.010.32-0.03-0.05-0.500.09-0.09-0.14S-0.39-0.72-1.00-0.84-0.72-0.64-0.65-0.75-1.43-0.20-1.08-0.21-1.07-0.58-0.64-0.73-0.15-0.46-0.58-1.13-0.27-1.43-0.20-0.640.00-0.01-0.04-0.040.060.070.06-0.26-0.20-0.05-0.060.14-0.03-0.08-0.03-0.05-0.04T-0.41-1.59-0.89-1.96-1.67-0.810.00-1.23-1.260.00-1.21-0.60-0.92-0.33-0.24-1.28-0.18-0.41-1.12-0.80-0.35-1.500.00-0.91-0.320.000.00-0.050.11 -0.03-0.01-0.38-0.20-0.02-0.100.14-0.09-0.310.21-0.06-0.08N-1.27-1.24-0.91-1.39-0.43-0.70-0.26-1.26-2.43-0.62-0.06-1.92-0.44-0.39-1.84-1.93-0.350.00-1.92-0.99-0.17-1.42-0.41-0.900.04-0.29-0.60-0.04-0.250.03-0.02-0.20-0.20-0.140.020.23-0.12-0.080.280.000.03Q-0.19-1.57-0.57-1.53-1.28-0.53-0.20NaNNaN0.09NaN-1.13-0.24-0.24-1.82-0.80-0.010.06-2.00-0.25-0.08-0.66-1.93-0.96-0.01-0.02-0.85-0.10-0.370.120.00-0.35-0.06-0.11 -0.040.200.03-0.130.19-0.07-0.04DNaN-1.520.03-1.57-1.21-1.58-0.84NaN-0.32-1.510.00-0.53-0.38-1.96-1.41-1.44-0.17-0.54-1.18-0.40-1.26-2.04NaN-2.28-0.32-0.35-0.72-0.08-0.340.020.11 -0.16-0.27-0.050.060.270.180.030.280.070.12E-1.44-1.270.00NaNNaN-1.27-0.82-1.35-0.91-0.70-0.16-0.890.00-0.26-0.53-1.400.00-0.35-0.490.00-0.49-1.20-1.01NaN-0.38-0.17-0.61-0.07-0.720.130.10-0.19-0.24-0.060.050.230.11 -0.230.270.00-0.01H-1.32-1.97-1.21-1.46-0.32-0.22-1.21-0.98-2.64-0.14-0.44-0.05-1.12-1.04-1.37-1.45-0.42-0.33-1.35-1.00-0.29-1.03NaN-0.35-0.06-0.09-1.00-0.10-0.44-0.05-0.27-0.48-0.27-0.08-0.130.08-0.03-0.240.19-0.15-0.11 K-1.41-1.35-0.41NaN-1.610.03-1.17-1.24-0.97-0.10-1.54-1.08-0.120.00-1.06-0.65-0.07-0.45-1.71-0.390.11 -1.54NaN-0.270.08-0.14-0.23-0.04-0.540.12-0.07-0.32-0.01-0.15-0.060.22-0.08-0.160.14-0.050.09R-0.87-1.57-1.72-1.74-1.610.00-1.38-0.28-1.87-0.29-1.35-1.59-1.13-0.13-1.530.00-0.07-0.33-2.09-0.500.00-1.34-2.030.000.04-0.23-0.60-0.26-0.650.01-0.33-0.46-0.18-0.43-0.140.00-0.43-0.360.07-0.17-0.10Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged AmiE I122L 123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960Mutation MRHGDISSSNDTVGVAVVNYKMPRLHTAAEVLDNARKIAEMIVGMKQGLPGMDLVVFPEYSTOP *-1.72-0.23-1.09-1.48-1.36-1.51NaN-0.20-0.16-1.44-1.61-2.35-1.34-1.10-1.59-1.47-1.78-1.43-1.39-0.52-0.64-1.86-1.34-1.52-1.11 -1.51-1.67-1.64-1.50-0.30-1.40-1.27-1.16-1.48-2.08-0.93-0.72-1.59NaN-0.57-1.67-1.23-1.48-2.47-1.55-0.51-0.71-1.70-1.42-1.73-1.74-1.56-1.62-1.11 -1.82-1.29-1.03NaN-0.25-0.10FNaNNaN-0.46NaN-1.64-0.46NaN-1.220.21-1.32-1.15-1.35NaN-1.35-1.35-1.79-1.59-1.39-1.87-0.29-1.80-1.48-1.12-1.40-0.25-0.88-0.93-1.23-0.60-1.27-0.72-0.68-0.68-1.42-1.66-0.80-0.60-0.50NaN-1.59-0.20-0.54-0.60-0.75-0.92-1.41-0.77-1.23-0.04-1.36-1.21-1.43-1.06-1.14-0.30-1.930.00-1.17-1.23-0.08WNaNNaNNaNNaN-1.77NaNNaN-0.21NaN-1.33-1.12NaN-1.09-0.85-1.64-0.95-1.14-1.44-1.15-0.61-1.89-3.09-1.39-3.41-1.70-0.81-1.82-1.22-0.64-1.70-1.08-1.65-0.54-1.80-1.31NaN-1.17-1.00-1.17-1.45-1.17-1.73-0.53-0.67-1.42-1.26-0.98-1.52-0.50-1.10-1.13-1.33-1.22-2.13NaNNaNNaNNaN-1.43NaNY-0.93NaN-0.23-1.87-0.57-1.45NaN-1.130.34-0.46-0.33-1.11 -1.76-2.08-1.83-1.41-1.50-2.18-0.460.00-1.49-1.23-1.71-1.08-1.20-0.18-0.78-0.75-0.52-1.18-1.28-0.72-0.32-1.11 -2.67-0.36-0.45-4.79NaN-1.06-0.93-1.36-0.34-0.74-1.46-1.76-0.59-0.94-0.01-1.12-1.47-1.60-0.54-1.55-1.79NaN-0.20NaN-1.520.00P-1.84-1.380.41-1.61-1.72-1.73NaN-0.57-0.360.03-1.65-0.93-1.46-1.22-1.28-1.19-1.49-1.54-1.89-1.35-0.10-0.980.00-1.23-1.32-0.69-1.040.10-0.35-1.28-1.43-0.32-1.21-2.09-0.74-0.56-1.34-1.38-0.38-1.73-1.34-1.69-0.87-1.14-1.47-1.39-1.00-1.67-0.390.00-1.56-1.36-1.59-0.65-1.22-1.43NaN0.00-1.54-2.35START M0.00NaNNaN-1.22-1.73-0.58NaN-1.440.06-0.27-2.54-0.66-1.00NaN-1.32-1.85-0.30-0.26-1.27-1.64-0.210.00-0.79-1.43-0.38NaN-0.82-0.35-0.23-0.49-1.430.03-0.29-1.35NaNNaN-0.41-0.97NaN-0.720.00-0.42-0.22-0.550.00-0.29-0.28-1.400.05-1.25-1.950.00-1.67-0.29-1.22-0.05NaN-0.85NaNNaNI-0.19-0.200.23-0.93-1.490.00-0.010.070.27-0.21-1.30-0.20-0.17-2.30-0.39-1.43-1.60-0.40-0.65-1.29-1.25-0.26-2.12-1.10-0.11 -1.14-0.72-0.74-0.35-1.63-0.260.09-0.40-0.78-1.60-0.85-0.430.00-1.06-1.65-0.150.000.03-1.85-0.31-1.67-0.59-1.42-0.13-1.04-1.50-0.16-1.60-0.78-0.19-0.03-0.15-1.47-1.12-0.40L-0.32-2.060.20-1.86-1.57-0.59NaN-0.36-0.14-0.26-1.64-1.34-0.45-1.33-0.61-1.29-1.12-0.75-1.46-1.45-1.13-0.69-0.37-1.010.00-0.56-0.92-0.68-0.43-1.17-1.140.00-0.33-1.53-1.26-0.30-0.51-0.64-1.50-1.15-0.25-0.40-0.30-0.79-0.09-1.87-0.51-1.440.00-0.53-1.830.24-1.420.00-0.59-0.45-0.06-0.28-1.44NaNV-0.29-1.270.16-0.29-0.59-0.13-1.22-0.36-0.38-0.35-0.58-1.530.00-0.330.00-0.130.000.00-1.59-1.45-0.66-0.61-0.98-0.75-0.17-1.31-1.03-0.12-0.25-0.380.00-0.16-0.16-1.69-0.15-0.59-0.48-0.25-0.10-0.62-0.250.090.00-0.65-0.45-1.71-0.39-1.02-0.52-0.95-0.66-0.37-0.56-0.840.000.00-0.23NaN-0.32NaNA-1.13NaN-0.270.08-0.78-1.45-0.660.050.180.17-1.25-0.16-0.33-0.54-0.540.00-0.56-0.61-1.55-1.22-0.28-1.73-0.97-0.78-0.52-1.05-0.390.000.00-1.01-0.62-0.43-0.05-1.180.00-0.20-0.15-1.620.00-0.13-1.26-1.12-0.33-0.10-1.08-0.89-0.10-0.39-1.13-0.75-1.23-1.07-1.82-1.82-0.26-0.31-1.24-0.42-1.22-1.22G-1.57-0.42-1.420.00-0.54-1.24-0.070.270.26-0.11 -0.52-0.60-0.750.00-0.83-0.96-0.85-0.48-1.32-1.67-0.59-2.43-1.91-0.93-1.33-1.23-0.28-0.09-0.04-0.30-0.81-0.72-0.15-1.08-0.60-0.29-0.56-1.64-0.06-0.21-1.12-1.92-0.420.00-1.80-2.05-0.600.00NaN-1.510.00-1.70-0.66-1.31-0.53-0.51-0.96-1.35-0.24NaNC-1.31-1.31-0.22-0.05-0.20-1.12-0.11 0.230.20-1.52-1.18-0.17-0.98-0.13-0.85-0.12-0.92-0.51-1.80-0.28-0.60-1.13-1.77-0.39-0.04-1.04-0.19-0.09-0.23-0.67-0.68-0.37-0.33-1.64-0.92-0.13-0.24-1.13NaN-0.62-0.63-0.82-0.15-0.17-1.19-1.36-0.44-0.55-0.62-1.01-0.55-0.40-1.88-1.97-0.63-0.54-0.33NaN-1.10-0.09S-1.35-0.430.040.06-1.48-0.990.000.000.00-0.22-1.42-0.20-1.47-0.38-1.41-0.48-1.55-1.41-0.74-1.71-0.26-1.42-0.22-0.41-0.50-0.530.040.120.04-0.56-1.40-0.68-0.15-0.46-0.43-0.08-0.21-0.88-0.15-0.14-1.30-0.91-0.45-0.14-1.54-1.06-0.20-1.10-1.28-0.27-0.90-2.26-1.44-1.65-1.28-1.23-0.20-0.22-1.71-0.84T-0.37-0.800.04-0.90-1.55-0.63-0.180.140.20-0.02-2.020.00-1.95-2.03-1.31-0.48-1.31-1.26-1.27-1.52-0.22-0.67-0.72-0.63-0.47-0.820.000.01-0.08-0.97-0.74-0.22-0.20-0.40-0.36-0.18-0.51-0.62-0.16-0.30-0.30-0.52-0.32-0.67-0.81-1.08-0.23-1.60-0.73-0.75-1.65-0.56-1.54-1.34-2.03-1.71-1.26-0.11 -1.37-2.06N-1.47-2.010.07-0.47-0.34-0.64-0.100.030.170.00-0.50-0.10-1.83-1.52-1.66-1.69-1.54-1.220.00-1.06-0.65-1.40-1.35-0.77-0.62-0.490.000.02-0.14-0.91-1.25-0.59-0.11 0.00-1.560.13-0.13-0.60NaN-0.01-1.90-0.60-0.52-0.12-1.37-0.55-0.29-1.53-1.62-1.39-1.75-2.42-0.53-1.36-2.13-1.00-1.57NaN-2.13-0.08Q-1.27-1.59-0.07-1.41-1.45-1.78NaN-1.120.120.19-1.51-0.62-2.37NaN-2.17-1.34-1.47-3.36-0.60-1.51-0.65-1.40-0.34-0.65-1.18-0.86-0.730.22-0.06-0.02-1.38-0.15-0.06-0.60-1.33-0.11 -0.22-1.77NaN-0.16-0.50-1.31-0.09-0.36-1.37-1.030.00-1.60-0.61-0.49-1.69-2.10-1.73-0.59-1.54NaNNaNNaN-0.29NaND-1.57-2.16-0.48-0.220.00-1.61NaN-0.850.41-0.060.00-1.90-1.11 -0.23-1.31-0.71-1.34-1.40-0.50-0.95-1.27-1.64-1.93-1.24-1.37-0.70-0.49-0.30-0.03-0.71-0.68-1.760.00-0.53-0.35-0.75-0.55-2.13-0.930.22NaN-3.10-0.42-0.43-1.93-2.17-0.65-0.82-1.50-1.37-0.28-1.320.00-2.06-0.17-1.90-0.96NaN-0.44-0.79E-1.38-1.76NaN-0.85-0.52-1.56NaN-1.13-0.21-0.12-0.46NaN-0.22-1.22-0.50-1.25-0.51-0.52-1.29-1.74-0.21-1.19-3.34-1.38-1.37NaN-0.82-0.52-0.090.00-1.44-0.800.00-0.73-1.85-0.21-0.23-1.35-0.080.00-1.39-1.82-0.10-0.71-1.50-0.37-0.35-1.88-2.08-1.17-2.37-1.44-0.22-1.46NaN-0.13NaN-0.850.00NaNHNaNNaN0.00-1.71-1.17-2.36NaN-1.550.26-0.26-0.85-1.16-1.55-1.17-1.80-1.52-2.18-2.21-1.11 -0.61-0.92-1.25-1.50-0.44-1.050.00-0.390.09-0.17-0.57-1.53-0.37-0.27-1.23-1.480.00-0.17-1.85-1.00-0.21-0.90-1.79-0.25-0.20-1.67-1.57-0.19-1.31-0.84-1.21-1.79-1.47-1.23-1.24-1.54-0.70-1.700.13NaN-0.46K-0.51-0.38-0.85NaN-1.44-1.34NaN0.14-1.000.03-1.07-1.47-1.26-1.45-1.64-1.72-1.47-1.42-0.50-1.750.00-0.90-1.31-0.36-1.45-0.46-0.310.43-0.07-0.32-1.730.02-0.30-0.58-1.980.120.00-1.52NaN-0.21-0.24-1.88-0.50-0.63-0.740.000.07-1.51-1.14-1.23-2.41-0.65-2.17-2.79NaNNaN-0.85NaN-0.17-1.43R-0.750.00-0.21-0.84-1.29-1.280.00-0.40-1.450.02-1.38-1.49-1.64-0.54-1.36-1.55-1.51-1.28-1.36-1.33-0.17-1.38-0.940.00-1.46-0.24-0.340.36-0.13-1.06-1.240.04-0.29-1.54-1.620.00-0.07-1.43-1.76-0.20-0.62-1.44-0.58-0.37-1.35-0.570.03-1.39-0.80-0.99-1.26-0.35-1.67-1.25-1.26-3.39-1.06-0.46-1.16-1.40AmiE I122L 616263646566676869707172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110 111112 113 114 115 116 117 118 119 120Mutation SLQGIMYDPAEMMETAVAIPGEETEIFSRACRKANVWGVFSLTGERHEEHPRKAPYNTLVSTOP *-1.17-1.03-0.49NaN-1.98-1.57-0.48-1.72-1.56-1.43-0.35-1.46-1.37-0.66-1.30-1.82-1.26-1.63-1.44-1.85-1.60-0.76-0.57-1.30-0.13-1.67-1.60-1.35-1.92-1.68-0.27-0.77-0.71-1.72-2.05-1.76-0.54-2.32-1.75-1.81-1.83-1.95-1.87-2.23-0.83-1.31-1.62-0.63-0.28-1.35-1.53-1.00-0.61-2.33NaNNaNNaNNaNNaNNaNF-0.18-0.36-1.55NaN-0.44-1.35-0.17-1.30-0.49-0.40-0.49-1.02-0.26-1.31-0.59-1.32-0.96-0.57-0.76-1.53-1.98-0.68-1.38-1.97-0.73-0.620.00-1.00-0.69-1.73-0.31-1.60-1.27-1.63-3.66-0.79-0.93-1.78-1.470.00-0.33-0.25-2.26-1.59-1.64-1.12-1.58-1.82-0.66-1.26-1.68-0.18-2.02-0.90NaNNaNNaNNaNNaNNaNWNaN-1.00-2.67-0.85-1.33NaN-0.66-1.30-0.38-0.44-0.76-1.24-0.72-1.56-1.22-1.32NaN-0.51-1.64-1.22-1.45-0.99-1.51-1.37-1.54-0.84-0.37NaN-0.06NaN-0.17-1.50-1.45-2.74NaN-1.200.00-1.50NaN-1.17NaN-1.81-3.53-1.80-2.07NaNNaN-1.71NaN-1.24NaN-0.59NaNNaNNaNNaNNaNNaNNaNNaNY-0.11 -1.70-2.15NaN-1.47-1.790.00-0.51-0.27-0.25-0.84-1.19-0.08-0.76-0.64-2.61NaN-0.25-1.38-1.20-1.57-0.50-1.57-1.49-1.00-1.52-0.31NaN-0.26-1.31-0.23-1.29-0.78-1.76-0.42-2.66-0.92-2.15NaN-0.51-0.77-1.30-1.73-1.75-1.59NaN-0.41-2.98-0.70-0.42-2.44-0.01-1.49-0.74NaNNaNNaNNaNNaNNaNP-0.26-1.55-1.09-1.53-0.97-0.43-1.05-1.440.00-0.82-1.09-1.73-1.87-1.27-1.15-1.00-1.24-0.17-1.530.00-1.620.34-1.33-1.38-1.54-1.95-1.91-1.33-0.89-1.01-1.35-1.62-1.18-1.50-1.89-2.15-1.91-2.11 -2.30-1.88-0.75-0.53-1.57-1.97-2.33-0.56-1.47-0.61-1.84-1.410.00-2.16-1.52-0.49NaNNaNNaNNaNNaNNaNSTART MNaN-0.76-1.45NaN-0.860.00-0.73-1.11 -0.33-0.12-0.230.000.00-0.39-0.30NaN-0.04-0.04-0.64-1.34-2.10-0.21-1.46-0.25-0.50-0.63NaN-1.000.21-1.17NaN-1.00-0.21-1.15-2.56NaN-1.45NaN-0.22NaN-1.58-0.18-1.66-3.28-4.70NaN-1.77-1.23-0.50-1.84-1.73-0.16-0.35-0.05NaNNaNNaNNaNNaNNaNI-1.03-0.60-1.42-1.820.00-0.17-0.69-1.12-0.75-0.05-0.48-0.28-0.09-0.85-0.60-1.72-0.64-0.400.00-1.50-1.44-0.04-1.490.17-1.060.00-0.27-0.36-0.54NaN-1.30-0.59-0.41-2.53-0.92-0.59-1.76-1.56-0.40-0.46-2.37-0.07-0.80-1.82-2.27-0.67-3.22-1.18-0.96-1.74-3.31-0.22-1.76-1.73NaNNaNNaNNaNNaNNaNL-1.090.00-0.88-1.33-0.56-0.52-0.49-1.18-0.51-0.10-0.27-0.43-0.03-0.64-0.23-1.45-0.33-0.20-0.67-0.44-1.55-0.15-1.43-0.29-0.68-0.77-0.05-1.28-0.26-2.47-1.77-1.07-0.33-1.64-1.86-0.94-0.51-1.74-0.54-0.39-2.310.00-2.03-1.66-1.64-0.44-0.73-1.57-0.42-0.77-0.80-0.09-2.22-0.80NaNNaNNaNNaNNaNNaNV-1.57-0.87-1.62-0.23-0.27-0.13-0.76-0.58-0.470.00-0.26-0.17-0.19-0.47-0.47-0.140.00-0.080.10-1.50-0.770.06-0.970.15-0.26-0.32-0.41-1.47-0.33-0.32-1.43-0.76-0.48-0.79-1.540.00-3.05-0.510.00-0.92-1.89-0.24-1.67-0.68-0.78-1.32-1.82-0.71-0.29-1.78-2.05-0.21-2.15-0.19NaNNaNNaNNaNNaNNaNA-0.34-1.73-1.55-0.83-0.67-0.45-0.91-0.77-0.170.00-1.21-0.64-0.980.05-0.410.00-0.200.00-0.34-0.83-1.250.12-0.45-0.74-0.02-1.47-1.490.350.310.00-1.58-0.430.030.00-1.12-0.70-1.69-0.84-0.51-2.46-1.00-0.94-0.64-1.14-1.58-0.73-2.65-0.88-0.48-1.98-1.10-0.09-1.360.00NaNNaNNaNNaNNaNNaNG-1.00-1.76-1.160.00-1.48-1.42-2.03-0.57-0.380.01-0.33-1.86-1.20-0.17-0.84-0.68-0.65-0.31-1.48-0.890.00-0.48-0.72-0.88-0.20-1.96NaN0.01-0.27-0.67-0.53-0.61-0.92-1.08-0.08-0.53-0.870.00-0.77-1.49-2.30-1.34-1.670.00-0.85-0.42-1.71-0.58-0.24-4.22-2.46-0.47-1.71-0.71NaNNaNNaNNaNNaNNaNC-0.27-0.94-1.88-0.17-0.51-0.70-0.08-0.59-0.46-0.13-0.45-0.27-0.96-0.38-0.19-0.44-0.400.080.15-1.45-0.21-0.28-1.30-0.59-0.63-1.06-0.22-0.20-0.06-1.270.00-0.81-0.52-0.88-0.830.02-0.45-0.43-1.34-0.51-1.11 -0.41-1.99-0.63-2.11 -0.15-3.50-1.35-0.26-2.15-1.40-0.31-1.22-0.51NaNNaNNaNNaNNaNNaNS0.00-0.48-0.75-0.54-0.77-0.48-0.56-0.41-0.17-0.01-0.44-1.20-1.06-0.01-0.48-0.35-1.430.16-0.94-0.53-0.70-0.26-1.25-0.77-0.54-1.51-0.270.000.06-0.40-0.60-0.55-0.17-0.77-0.50-1.26-1.30-0.84-1.61-0.590.00-1.67-0.92-0.54-2.01-0.28-1.74-1.23-0.65-2.00-0.83-0.18-1.51-0.10NaNNaNNaNNaNNaNNaNT-0.340.09-0.77-1.56-0.76-0.34-0.51-0.66-0.120.02-0.37-0.62-0.45-0.170.00-0.64-1.350.05-0.79-0.55-1.59-0.21-1.430.00-0.68-0.61-1.60-0.61-0.06-0.42-1.49-1.01-0.04-0.66-0.66-0.30-2.27-1.66-1.80-1.78-0.93-1.440.00-1.82-2.01-1.45-2.27-1.64-0.91-1.71-0.78-0.15-0.94-0.10NaNNaNNaNNaNNaNNaNN-1.06-1.09-1.76NaN-0.59-0.51-0.26-0.11 -0.120.06-0.38-1.58-0.970.06-0.51-1.67-1.470.00-0.90-1.31-1.83-0.30-1.38-1.78-0.45-0.81NaN-0.300.16-1.78-2.00-0.83-0.25-1.210.00-1.47-1.36-1.49-1.44-1.61-1.86-2.49-0.98-2.37-2.05NaN-0.38-1.40-0.39-0.54-2.740.05-0.60-0.03NaNNaNNaNNaNNaNNaNQ-1.08-1.250.00-1.73-1.12-0.19-0.87-0.730.040.12-0.12-0.59-0.970.05-1.07-1.62-0.900.04-1.45-0.13-1.690.08-0.61-1.29-0.16-1.07NaN-1.190.28-2.05-2.22-0.180.35NaN-0.40-5.01-1.26-3.41-1.74-1.84-1.96-0.57-1.75-2.00-1.04-0.13-0.49-0.69-0.18-0.33-1.63-0.10-1.26-0.22NaNNaNNaNNaNNaNNaND-0.59-1.18-1.39-0.30-1.45-1.19-0.560.00-0.61-0.04-0.22-1.59-1.730.03-1.31-0.35-1.73-0.45-1.30-0.95-0.67-0.22-0.15-1.53-0.40-1.64-0.71NaN0.30-0.36NaN-1.280.21-1.76-0.36-0.61-1.90-0.27-1.74-1.38-1.88NaN-2.15-0.20-0.93NaN-0.89-0.48-0.01-1.04-1.53-0.32-1.67-1.28NaNNaNNaNNaNNaNNaNENaN-1.02-0.93-1.13-1.61-1.60-0.76-0.23-0.380.010.00-1.82-1.040.00-1.43-1.64-0.160.05-1.40-1.48-1.520.000.00-1.510.00-1.28-1.27NaN0.36-1.33-1.00-0.850.27-0.67-1.06-1.63-1.69-1.57-0.45-1.73-3.91-1.41-2.35-3.960.00NaN-1.700.000.00-1.67NaN-0.32-0.24-0.24NaNNaNNaNNaNNaNNaNHNaN-1.03-0.21-1.57-1.39-0.58-0.10-0.600.040.04-0.42-1.28-0.91-0.16-0.52-1.35-1.90-0.13-1.40-1.19-1.76-0.26-0.56-1.51-0.50-1.30-1.22-2.45-0.01-1.54-1.86-1.12-0.40-2.12-0.64-1.61-1.44-2.09-1.84-1.58-1.61-2.04-1.81-1.74-1.71-0.080.00-1.37-0.270.00-0.490.09-2.07-0.42NaNNaNNaNNaNNaNNaNKNaN-1.71-0.98-1.43-1.43-0.25-1.10-0.550.040.20-0.25-0.56-0.30-0.04-0.37-1.53-1.120.00-1.41-1.41-1.900.09-0.75-0.76-0.15-1.76-1.00-1.000.08-2.29-1.450.080.00-1.47-0.27-1.41-1.34-2.28-2.53NaN-2.59-2.63-1.96-1.74-0.63-0.28-2.03-0.56-0.32-1.62-2.030.060.00-0.48NaNNaNNaNNaNNaNNaNR-1.39-1.37-0.84-0.97-1.46-0.53-0.60-0.860.050.07-0.37-0.21-0.440.00-0.31-1.64-1.86-0.14-1.50-0.77-1.250.09-1.47-1.17-0.33-1.80-1.67-0.230.00-1.67-0.420.00-0.09-2.09-0.73-2.09-0.69-1.41-5.74-2.58-1.72-0.96-1.88-1.45-2.500.00-0.67-1.35-0.95-0.71-1.460.00-0.50-0.08NaNNaNNaNNaNNaNNaNAmiE I122L 121122123124125126127128129130131132133134135136137138139140141142143144145146147148149150151152153154155156157158159160161162163164165166167168169170171172173174175176177178179180Mutation LLDNNGEIVQKYRKIIPWCPIEGWYPGGQTYVSEGPKGMKISLIICDDGNYPEIWRDCAMSTOP *NaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.96NaN-0.37-0.70-2.37-1.71-1.61-0.44-0.58NaN-2.57-0.94-1.65-0.13-0.72-3.50-2.00-2.19-0.21-1.94-1.01-1.58-1.82-0.48-1.96NaN-0.46NaNNaN-0.61-1.79-0.70-0.64-1.93-2.01-0.60-1.34-1.93-1.77-1.78-0.36-1.93-0.34-1.40-0.17-0.38-1.25-0.13-2.68-1.36FNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-2.08NaN-1.21-2.25-0.340.09-1.91-0.68-0.63NaN-0.30-2.03-0.13-0.77-0.45-1.52-1.95-1.30-0.94-1.520.06-1.33-0.68-0.93-2.00NaNNaNNaNNaN-1.00-0.62-1.84-0.35-0.30-0.35-0.64-1.68-2.22NaN-1.12-0.25-2.39NaN-0.35-1.76-1.88-1.90-0.39-1.43-1.13WNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-2.64NaNNaNNaN-1.23-1.49-1.230.00-0.41NaN-1.29NaN-0.560.00-2.22-1.39-1.60NaNNaN-3.03-0.84-1.78-1.65-1.22-1.49NaNNaNNaNNaN-1.58NaN-1.32NaN-1.31-1.39-0.55-1.50NaN-2.29-1.06-0.74-2.07-1.15NaN0.00-1.11 NaN-0.17NaN-0.68YNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-2.37NaN-2.69-1.80-1.70-0.53-2.11 -0.51-0.50NaN-0.85-1.49-0.28NaN0.00-1.70-2.58-1.88-0.82-2.160.00-1.72-0.67-0.57-3.02NaN-2.65-1.17-0.74-1.11 -1.75-1.45-3.96-2.56-1.91-0.57-0.37-0.34-2.39-0.580.00-1.36-0.72-2.02NaN-1.82-0.24-0.06NaN-0.85PNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-2.01NaN-1.70-2.07-2.00-1.730.00-2.24-1.820.00-1.57-1.490.03-1.31-1.850.00-1.69-1.82-1.27-1.48-2.13-2.02-0.89-0.24-1.850.00-2.41-2.22-1.59-1.73-1.79-0.78-1.53-1.46-1.77-1.51-1.60-1.74-1.80-1.67-1.340.00-1.61-1.39-0.99-1.54-1.35-1.45-0.68-1.87START MNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.61NaNNaN-0.45-0.170.18-1.40-1.63-1.63NaN-0.58NaN-0.10NaN-1.48-1.35NaN-0.15-0.25NaN-1.20-1.93-2.05-0.42-1.24NaN-0.86NaN0.00-0.28-0.24NaN-0.07-1.16-0.40-1.60-2.19NaN-1.88-1.54-0.95-1.10-1.43-0.18-1.49-1.59NaNNaNNaN0.00INaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.40NaN-0.63-3.230.000.00-2.14-2.07-1.70NaN0.00-2.57-0.32NaN-1.95-1.42-1.92-0.77-1.20-0.66-1.76-0.69-1.86-0.08-1.72NaN-0.48-2.22-0.07-1.180.00-1.45-0.620.000.00-2.34-2.05-1.63-1.60-0.21-1.68-1.29-1.620.00-1.06-0.61-1.58-1.54-1.97-0.27LNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.33NaN-2.16-1.85-0.11 0.16-0.81-1.01-1.78-0.16-0.46-1.77-0.25-0.19-2.14-0.50-1.63-0.55-0.36-1.89-1.51-1.40-1.94-0.25-2.00-0.18-1.77-2.24-0.07-0.66-0.41-0.780.00-0.99-0.41-1.99-3.07-2.58-1.94-1.83-1.48-0.42-1.68-0.84-0.19-1.64-2.71-1.50-1.33-0.41VNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.26NaN-2.11 -2.23-0.540.05-1.96-1.73-0.68-1.760.09-0.71-0.17NaN-2.05-1.75-0.82-0.19-0.72-1.17-1.860.00-2.530.02-1.24NaN-1.88-0.30-0.18-1.25-0.17-1.94-0.96-0.300.10-1.94-0.52-0.20-0.92NaN-1.90-1.65-0.260.02-1.02-1.51-0.16-1.49-0.05-0.29ANaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.74NaN-1.83-2.08-2.34-0.41-1.19-1.31-0.57-0.40-0.72-1.80-0.04NaN-2.76-0.96-1.380.02-0.16-1.00-2.27-0.71-0.90-0.20-1.68-0.49-1.49-0.60-1.31-1.04-1.86-0.68-1.52-2.24-0.70-1.66-1.16-1.40-1.41-1.70-1.58-0.91-0.14-1.68-1.80-2.03-0.56-1.680.00-0.82GNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.72NaN-0.36-3.19-1.74-1.55-2.15-1.42-0.61-1.15-1.39-0.570.00NaN-1.64-1.700.000.00-0.43-3.04-1.95-1.07-1.60-0.410.00-1.73-2.450.00NaN-1.61-1.70-1.86-1.47-1.37-2.80-1.12-0.51-0.300.00-2.54-1.35-1.43-0.24-1.30-0.41-0.22-0.12-0.26-0.32-1.21CNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.43NaNNaN-1.47-2.460.04-3.31-0.370.00NaN-0.97-9.04-0.07-0.51-0.49-2.63-0.47-0.11 -0.07-2.93-0.42-0.27-0.59-0.29-0.77-1.73-2.05-0.20NaN-1.02-1.15-1.68-0.41-1.190.390.00-1.18NaN-0.48-1.20-0.18-1.25NaN-0.87-0.07-1.69-1.330.00-1.13-1.65SNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.81NaN-0.88-1.73-1.53-0.39-0.86-1.41-0.54-0.23-0.69-2.150.06-0.29-1.00-0.60-0.940.10-0.09-0.92-1.48-1.840.00-0.18-1.33-0.35-2.05-0.31-1.59-1.460.050.00-0.65-0.86-0.74-0.80-1.41-1.76-0.54-0.520.40-0.47-1.44-0.91-0.53-0.41-1.89-0.24-0.47-1.19TNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.07NaN-0.95-1.50-0.77-0.87-0.69-1.610.01-0.35-0.40-2.22-0.04NaN-0.56-0.73-1.850.14-0.100.00-1.45-1.40-0.050.05-1.61-0.53-2.79-1.24-0.22-1.20-0.65-0.93-1.43-0.73-0.18-2.15-1.47-2.25-2.19-1.04-1.42-0.25-2.39-0.25-2.29-1.26NaN-1.27-0.26-0.45NNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.97NaN-2.42-0.63-0.62-0.14-1.71-3.33-1.18NaN-0.54-2.630.03NaN-0.58-1.59-1.220.25-0.03-2.22-0.96-2.26-0.71-0.04-1.91NaN-0.31NaN-1.09-0.76-0.81-2.29-1.66-0.41-0.45-2.67-0.31-0.50-1.680.00-0.72-1.37-1.06-0.54NaN-1.34-0.44NaN-1.88-1.83QNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.89NaN-1.51-1.16-1.39-1.46-0.54-1.73-2.02-0.21-0.64-0.960.14NaN-2.96-0.44-2.080.010.00-1.90-1.95-1.76-2.57-0.16-1.99-0.23NaNNaNNaN-0.72-1.33-1.93-1.77NaN-1.50-1.73-1.70NaN-1.91-2.07-2.76-0.31-0.60-0.97-1.24-1.58-2.62-0.81-1.57-1.87DNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-3.46NaN-1.33-2.40-2.65-1.94-1.99-2.88-0.58NaN-2.80-0.43-0.45NaN-0.97-1.97-0.680.00-0.55-2.24-1.22-0.80-1.090.05-0.71NaN-2.05-0.12NaN-1.78-1.59-1.47-3.00-1.31-1.88-2.270.000.00-0.69-0.24-0.77-1.70-0.37-1.05-1.07-1.830.00-0.76-0.21-1.54ENaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.60NaN-1.78-0.32-1.27-1.94-1.96-2.08-1.51NaN-3.190.00-0.45NaN-4.06-2.31-1.950.07-0.10-2.25-1.47-1.71-7.100.00-2.47-1.31-0.41NaNNaN-0.44-1.60-1.75-1.85NaN-1.57NaN-1.87-0.40-1.68-0.87-2.23-1.030.00-1.42NaN-1.12-0.20NaN-2.67-1.85HNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.82NaN-1.03-1.71-1.92-0.22-1.80-1.52-1.62-1.95-0.52-2.460.01NaN-0.48-1.93-1.62-0.03-0.16-1.68-0.48-2.13-2.12-0.29-1.83-2.17-2.11 -1.60-0.75-1.21-1.49-2.14-2.07-1.29-1.30-1.83-0.85-0.68-2.21-1.49-0.29-1.46-1.19NaN-1.49-2.00-0.53-1.19NaN-2.95KNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN0.00NaN-0.550.00-3.05-2.39-1.53-2.04-3.58-1.22-0.67-0.64-0.18NaN-1.71-2.07-1.400.11 -0.16-1.03-2.54-1.64-1.71-0.22-1.62-1.810.00-1.31-0.190.00-2.26-1.91-1.90-2.02-1.67-1.67NaN-1.57-2.01-0.13-1.15-1.12-0.23-2.62-1.78-0.72NaN-1.22-1.50-0.53RNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.15NaN0.00-0.70-1.82-1.72-1.30-0.83-0.77-0.36-1.08-2.02-0.13-0.10-2.16-0.84-1.92-0.09-0.36-1.32-1.88-1.70-1.79-0.27-1.72-0.77-0.53-0.63-0.68-0.26-1.82-1.71-1.70-2.60-2.29-0.72-2.09-3.85-1.38-2.39-1.45-1.03-1.70-1.78-0.270.00-1.70-0.18-1.69-0.54AmiE I122L 181182183184185186187188189190191192193194195196197198199200201202203204205206207208209210211 212213214215216217218219220221222223224225226227228229230231232233234235236237238239240Mutation KGAELIVRCQGYMYPAKDQQVMMAKAMAWANNCYVAVANAAGFDGVYSYFGHSAIIGFDGSTOP *-0.59-1.59-1.76-0.29-1.05-1.34-1.25-2.24-0.52-0.08-1.00-0.54-1.95-0.25NaN-2.82-0.39-1.71-0.35-0.77-2.48-1.85-1.57-3.25-0.56-2.26-1.49-1.74-0.15-1.41-2.77-1.46-0.35-0.48-1.80-1.21-1.59NaNNaN-0.84-2.20-1.99-1.58-1.32-1.54-2.14-1.00-1.42-0.30-2.11 NaN-1.86-1.54-1.57-1.55-1.57-1.80-1.77NaN-1.40F-1.27-1.44-1.57NaN-0.50-0.26-0.55-1.15-0.34-1.11 -1.76-0.08-2.19-0.39-1.63-1.34-1.55-0.68-1.37-1.52-0.46-0.90-1.27-2.31-1.46-1.84-1.51NaN-0.46-1.52-1.81-1.21-0.31-0.43-0.59-1.70-1.59-1.46-1.49NaN-1.65-2.440.00-1.49-1.43-0.21-0.03NaN-0.040.00NaN-1.25-2.21-1.33-0.68-0.26-1.270.00-1.15-1.39W-1.45-2.57-1.25NaN-0.79-1.41-2.05NaN-0.44NaN-1.38-1.56-1.69-1.38-1.29NaN-0.62-1.16NaN-1.99-0.70-0.56-1.24NaNNaN-0.96-1.98NaN0.00-2.02NaN-1.46-0.68NaN-1.30NaN-1.61-1.15-0.98NaNNaN-1.42-0.28-0.84NaNNaNNaNNaNNaN-0.34NaNNaN-1.02-1.35-2.00-1.83-1.49NaNNaN-1.00Y-1.73-1.67-1.47-1.30-1.97-1.46-2.18-1.65-0.10NaN-1.320.00-1.050.00-0.98-1.70NaN-0.38-1.23-2.30-0.63-0.87-1.46-2.54-1.54-2.94-1.30NaN-0.54-1.47-0.41-0.61-0.350.00-1.50-1.76-2.17-2.38-0.35NaN-1.00-1.74-0.18-0.25NaN-0.960.00-0.910.00-0.03NaN-0.17-1.58-1.88-1.650.21-2.26-0.39-0.24-1.90P-1.51-1.66-1.31-2.34-1.80-1.61-1.50-0.92-0.92-0.46-1.82-1.37-0.08-1.600.00-0.89-1.87-0.88-0.41-1.06-1.61-1.40-1.60-0.75-1.10-0.79-2.06-0.59-1.76-1.16-1.83-1.38-1.49-1.72-1.67-0.93-1.79-0.76-1.15-0.54-0.79-1.51-1.61-1.47-1.71-1.20-2.19-1.51-1.16-1.23-1.12-0.88-1.61-1.17-1.83-1.61-1.59-1.60-1.48-1.70START M-0.44-1.59-2.00NaN-0.69-0.44-1.83NaN-1.51NaN-0.93-1.570.00-1.15NaN-1.17-0.40-0.76-0.58-1.280.310.000.00-1.41-0.01-1.190.00-0.98-1.30-1.97-1.94-0.37-1.06-1.40-1.17NaN-1.50NaN-1.13NaNNaN-1.20-1.23NaNNaN-0.66-0.74-1.07NaN-0.74NaN-0.95-2.260.19-6.33-0.80-2.21-1.03NaN-1.86I-1.63-1.53-3.68-1.76-0.950.00-0.06NaN-1.34-1.54-2.02-2.10-0.35-1.82-1.08-1.72-0.45-0.98-0.36-1.500.20-0.07-0.26-1.53-1.35-0.88-0.19-2.02-1.11 -1.20-0.45-0.37-0.86-1.25-0.40-1.56-0.33-1.58-0.39-0.81-1.44-1.56-1.19-1.97NaN-0.10-1.82-0.33-1.61-0.52NaN-1.28-0.67-0.550.000.00-1.80-0.94-1.28-2.27L-1.47-1.72-1.78-1.610.00-0.90-0.94-0.47-1.75-0.18-1.76-1.80-0.66-1.31-0.36-1.34-0.91-0.96-0.54-1.180.17-0.08-0.30-1.75-1.76-1.52-0.66NaN-0.37-1.60-1.77-0.70-1.25-1.71-0.96-1.36-0.56-3.12-1.66-1.51-1.74-1.30-0.66-1.70-2.51-0.49-1.17-1.83-2.39-0.53-1.94-0.52-2.51-0.42-0.57-1.12-1.96-0.51-1.37-1.57V-1.68-0.83-0.19-0.21-0.93-0.270.00-1.46-1.29-1.11 -0.23-1.75-0.42-2.99-1.26-0.15-0.87-0.49-0.34-1.460.00-0.19-0.25-0.29-1.35-0.08-0.44-0.12-1.66-0.32-1.63-0.370.03-1.780.00-0.400.00-0.17-1.63-0.15-0.23-1.02-1.10-0.24-0.210.00-1.56NaN-0.86-0.78-0.63-3.13-1.43-0.34-0.53-0.52-0.57-0.53-0.09-0.44A-1.33-1.310.00-1.21-1.46-2.10-0.46-1.24-0.080.07-0.51-1.39-0.71-0.16-0.580.00-1.03-0.71-0.18-1.00-0.07-0.09-1.290.00-2.380.00-1.930.00-1.900.00-1.70-0.95-0.52-1.59-0.570.00-0.540.00-1.260.000.00-1.58-1.31-1.13-0.74-0.26-0.87-0.25-1.09-1.66-0.41-1.23-1.510.00-1.66-1.82-0.59-2.04-0.61-0.94G-1.000.00-1.10-0.27-1.53-1.70-0.70-0.41-0.81-0.960.00-1.35-0.47-1.93-2.07-0.34-1.60-0.63-1.43-1.46-0.16-0.38-1.09-0.61-1.86-0.61-1.51-0.36-0.83-1.05-1.47-0.62-0.50-1.82-0.86-0.83-0.98-0.69-2.78-0.24-0.640.00-1.52-0.540.000.02-1.32-0.16-1.78-2.070.00-0.65-0.97-0.65-1.51-1.530.00-1.54-0.160.00C-1.39-0.59-1.15-1.84-1.09-2.05-0.39-0.200.00-0.22-0.21-0.39-0.46-0.28-1.540.04-0.81-1.00-0.81-1.550.07-0.15-2.230.10-0.930.13-1.18-1.74-0.11 -0.32-3.78-0.330.00-0.32-1.46-0.81-0.17NaNNaNNaN-0.84-0.62-0.53-0.86-0.18-0.20-0.04-0.12-0.02-0.31-0.25-0.76-0.440.14-1.48-1.44-0.43-0.22-1.04-0.26S-1.21-1.14-0.64-1.68-0.78-0.82-1.64-0.500.01-0.06-0.37-1.32-0.78-0.16-0.37-0.18-0.57-0.58-0.77-1.180.02-0.40-1.970.16-1.14-0.08-2.07-0.35-0.71-0.83-0.69-0.31-0.35-1.60-1.93-0.61-1.03-0.29-0.48-0.200.00-1.24-0.62-1.45-0.22-0.65-0.840.00-1.34-0.15-0.05-1.430.00-0.04-3.08-0.99-0.19-0.52-1.84-0.44T-1.10-1.56-0.50-1.68-2.17-0.54-1.95-1.77-1.74-0.37-1.08-1.52-0.46-0.99-0.24-0.30-0.97-0.68-0.45-1.410.13-0.03-0.50-0.08-0.73-0.01-0.49-0.22-1.68-0.53-1.28-0.24-0.15-1.45-1.49-0.23-0.39-0.21-0.55-0.190.10-1.88-0.95-1.46-1.33-0.09-1.73-0.29-0.92-0.41-1.72-1.20-1.27-0.08-0.99-0.99-1.38-1.36-1.61-1.54N-0.56-1.55-2.63-1.19-1.82-0.35-1.79-1.45-1.93-0.66-1.90-0.80-0.85-0.09-1.49-0.48-0.47-0.40-1.29-0.76-0.07-0.01NaN-0.16-0.18-1.20-1.06-1.51-2.09-2.140.000.00-1.55-1.04-1.59-1.57-1.72-1.480.00NaN-1.89-1.59-0.47-0.35-1.82-0.97-0.53-0.28-0.26-1.92-0.84-0.17-0.68-1.21-0.78-0.75-1.03-1.77-0.23-1.27Q-0.67-1.49NaN-0.45-1.90-1.64-1.75-1.42-1.690.00-1.19-1.63-1.15-0.04NaN-0.86-0.48-0.550.000.000.09-0.29-1.12-0.76-0.74-1.56-1.43NaN-1.70-1.63-1.430.12NaN-1.73-1.82-1.75-0.34-1.56-1.30NaN-1.90-3.15-1.06-2.17-0.97-0.66-1.84-1.30-1.35NaNNaN-0.50-1.570.14-1.75-1.81-1.45-1.15NaN-1.60D-1.66-0.64-1.32-0.26-5.05-1.43-0.65-1.47-2.08-1.19-0.14-0.84-2.57-0.92-1.39-0.43-1.190.00-1.87-1.44-0.49-1.40-1.82-0.45-1.56-2.29-2.33-0.18-1.28-1.50-0.40-0.59-0.98-4.36-0.42-0.51-1.75-2.02-0.23-0.16-1.23-0.87-1.910.00-0.22-0.31-0.62-1.16-0.29-1.14-0.05-0.54-1.70-0.76-1.53-2.02-0.46-1.670.00-0.30E-0.55-1.69-0.480.00-1.18-1.03-1.15NaN-1.37-0.07NaN-1.85-2.36-1.19NaN-1.27-0.260.11 -0.52-0.96-0.70-0.88-1.42-1.36-0.15-0.56-2.42NaN-1.82-0.48-1.63-0.82NaN-1.63-1.30NaN-0.61-0.32-0.60NaN-0.42-1.90-1.90-0.30-1.03-2.05NaN-1.54NaN-1.78NaN-1.40-1.86-0.99-1.39-1.31-1.20-2.14-0.20-1.10H-1.23-1.54-2.21-1.85-1.52-2.41-2.15-0.24-1.35-0.07-1.22-0.49-2.17-0.47-0.17-1.77-1.47-0.57-0.34-0.78-0.42-0.62-1.57-1.21-0.74-1.64-1.57-1.37NaN-1.24-1.40-0.58-1.60-0.48-1.47-1.57-1.87NaN-0.71NaN-1.69-1.69-0.21-0.60NaN-0.51-0.39-0.33-0.71-0.64NaN0.00-1.55-0.58-1.64-1.15-2.12-0.90-0.57-1.47K0.00-1.57-1.55-0.27-1.79-1.60-1.69NaN-1.65-0.20-1.13-1.53-0.39-1.23NaN-1.160.00-0.79-0.34-0.83-0.27-0.74-0.36-1.080.00-1.63-0.40-1.84-1.52-1.89-0.46-0.60-1.33-3.02-2.09NaN-1.33-2.26-0.32NaN-1.44-1.28-1.49-1.52-1.74-0.58NaN-0.45-0.93-1.25NaN-0.79-1.33-1.40-2.17-1.37-1.51NaNNaN-1.46R-0.28-1.30-1.93-1.35-1.85-1.58-2.610.00-0.43-0.10-0.83-1.36-0.93-1.18-0.62-1.64-0.06-1.02-0.47-0.810.09-0.35-0.84-1.76-0.11 -1.45-1.04-1.04-0.39-1.61-1.41-1.50-0.45-1.66-1.62-1.92-2.16-1.47-2.04-1.73-1.43-1.29-1.11 -1.53-0.68-0.36-1.61-0.24-2.60-1.23-0.58-1.02-0.74-1.35-2.59-1.57-1.07-1.95-1.70-1.24AmiE I122L 241242243244245246247248249250251252253254255256257258259260261262263264265266267268269270271272273274275276277278279280281282283284285286287288289290291292293294295296297298299300Mutation RTLGECGEEEMGIQYAQLSLSQIRDARANDQSQNHLFKILHRGYSGLQASGDGDRGLAECSTOP *-1.40-1.38-1.39-1.24-0.52-0.50-1.71-0.44-0.64-0.53-1.00-1.39-1.58-0.63-0.67-1.40-0.55-0.63-1.42-1.93-0.58-0.40NaN-1.44-1.10-1.93-2.78-1.30-1.59-1.94-0.35-1.33-0.24-1.26-2.83-0.58-1.48-0.39-2.15-0.50-1.57-0.90-1.51-0.84-0.97-2.27-0.79-0.29-1.65-0.62-4.11 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-1.29-1.39-1.46-0.59-0.60-1.40-1.04-0.82-1.50NaN-1.17-0.10-0.27-1.30-0.28-0.79-0.24-0.44-0.40-0.71-0.04-0.27-0.10-0.12-0.400.00-0.24-0.25P0.00-1.63-1.66-1.90-1.90-1.26-1.13-1.49-1.49-1.82-1.170.25-1.11 -1.33-0.99-1.84-1.44-1.67-1.39-1.20-1.21-0.76-1.98-1.16-0.08-0.71-0.08-0.45-0.55-0.07-0.29-0.520.00-0.450.060.21-0.030.000.210.070.01START MNaN-2.32-1.43-0.94-1.17-2.01-0.07NaN-1.030.01NaN-1.17NaN-0.26-1.57-1.21-0.23-0.04-0.19-0.67-0.180.00-0.81-0.52-0.07-0.48-0.51-0.20-0.68-0.18-0.24-0.79-0.62-0.28-0.11 0.04-0.06-0.350.14-0.10-0.17INaN-0.74-1.34-0.66-1.41-0.85-0.25NaN-0.13-0.10-1.06-1.29-1.28-1.01-1.78-0.45-0.35-0.42-0.53-0.70-0.310.17-0.26-1.19-0.26-0.34-0.19-0.35-0.31-0.23-0.31-0.68-1.030.13-0.240.03-0.08-0.520.19-0.02-0.10L-0.48-0.62-1.26-0.49-1.41-0.92-0.05-0.23-1.230.08-1.94-2.12-1.18-0.36-1.59-1.38-0.14-0.42-0.82-0.41-0.630.00-0.94-0.71-0.30-0.89-0.41-0.36-0.54-0.21-0.37-0.58-0.41-0.13-0.15-0.040.00-0.140.170.05-0.05V-1.80-2.44-0.43-1.11 -1.18-0.74-0.29-0.940.00-0.45-0.38-0.37-0.29-0.55-0.28-1.08-0.35-0.880.00-0.31-0.54-0.41NaN-1.46-0.11 -0.27-0.13-0.490.00-0.12-0.12-0.64-0.640.00-0.130.02-0.04-0.310.14-0.09-0.12A-0.63-2.03-0.97-1.90-2.04-1.46-0.67-1.27-0.49-0.42-0.840.00-0.27-0.190.00-1.58-0.01-0.22-0.32-0.39-0.04-1.53-0.51-1.16-0.15-0.27-0.09-0.14-0.020.000.06-0.33-0.23-0.04-0.040.05-0.01-0.280.13-0.13-0.08GNaN-1.60-0.29-1.80-2.30-0.89-1.75-1.07-0.68-0.87-0.30-1.02-0.18-0.99-0.72-0.64-0.44-0.77-0.67-1.19-0.50-1.30NaN-0.45-0.01-0.35-0.500.00-0.260.02-0.06-0.33-0.39-0.210.000.180.05-0.150.22-0.040.00CNaN-0.76-2.17-0.93-0.69-0.26-0.74-0.13-1.32-0.40-1.17-0.09NaN-0.86-0.04-1.46-0.55-0.61-0.35-0.35-0.22-1.49-0.07-0.37-0.12-0.28-0.13-0.30-0.12-0.11 -0.010.00-0.300.010.32-0.03-0.05-0.500.09-0.09-0.14S-0.39-0.72-1.00-0.84-0.72-0.64-0.65-0.75-1.43-0.20-1.08-0.21-1.07-0.58-0.64-0.73-0.15-0.46-0.58-1.13-0.27-1.43-0.20-0.640.00-0.01-0.04-0.040.060.070.06-0.26-0.20-0.05-0.060.14-0.03-0.08-0.03-0.05-0.04T-0.41-1.59-0.89-1.96-1.67-0.810.00-1.23-1.260.00-1.21-0.60-0.92-0.33-0.24-1.28-0.18-0.41-1.12-0.80-0.35-1.500.00-0.91-0.320.000.00-0.050.11 -0.03-0.01-0.38-0.20-0.02-0.100.14-0.09-0.310.21-0.06-0.08N-1.27-1.24-0.91-1.39-0.43-0.70-0.26-1.26-2.43-0.62-0.06-1.92-0.44-0.39-1.84-1.93-0.350.00-1.92-0.99-0.17-1.42-0.41-0.900.04-0.29-0.60-0.04-0.250.03-0.02-0.20-0.20-0.140.020.23-0.12-0.080.280.000.03Q-0.19-1.57-0.57-1.53-1.28-0.53-0.20NaNNaN0.09NaN-1.13-0.24-0.24-1.82-0.80-0.010.06-2.00-0.25-0.08-0.66-1.93-0.96-0.01-0.02-0.85-0.10-0.370.120.00-0.35-0.06-0.11 -0.040.200.03-0.130.19-0.07-0.04DNaN-1.520.03-1.57-1.21-1.58-0.84NaN-0.32-1.510.00-0.53-0.38-1.96-1.41-1.44-0.17-0.54-1.18-0.40-1.26-2.04NaN-2.28-0.32-0.35-0.72-0.08-0.340.020.11 -0.16-0.27-0.050.060.270.180.030.280.070.12E-1.44-1.270.00NaNNaN-1.27-0.82-1.35-0.91-0.70-0.16-0.890.00-0.26-0.53-1.400.00-0.35-0.490.00-0.49-1.20-1.01NaN-0.38-0.17-0.61-0.07-0.720.130.10-0.19-0.24-0.060.050.230.11 -0.230.270.00-0.01H-1.32-1.97-1.21-1.46-0.32-0.22-1.21-0.98-2.64-0.14-0.44-0.05-1.12-1.04-1.37-1.45-0.42-0.33-1.35-1.00-0.29-1.03NaN-0.35-0.06-0.09-1.00-0.10-0.44-0.05-0.27-0.48-0.27-0.08-0.130.08-0.03-0.240.19-0.15-0.11 K-1.41-1.35-0.41NaN-1.610.03-1.17-1.24-0.97-0.10-1.54-1.08-0.120.00-1.06-0.65-0.07-0.45-1.71-0.390.11 -1.54NaN-0.270.08-0.14-0.23-0.04-0.540.12-0.07-0.32-0.01-0.15-0.060.22-0.08-0.160.14-0.050.09R-0.87-1.57-1.72-1.74-1.610.00-1.38-0.28-1.87-0.29-1.35-1.59-1.13-0.13-1.530.00-0.07-0.33-2.09-0.500.00-1.34-2.030.000.04-0.23-0.60-0.26-0.650.01-0.33-0.46-0.18-0.43-0.140.00-0.43-0.360.07-0.17-0.10Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged ! 121 Figure A 20: AmiE I38V heatmap. AmiE I38V 123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960Mutation MRHGDISSSNDTVGVAVVNYKMPRLHTAAEVLDNARKVAEMIVGMKQGLPGMDLVVFPEYSTOP *-1.16-0.16-1.42-1.15-1.09-1.28NaN-0.27-0.09-1.18-1.38-1.12-1.38-0.88-1.40-1.16-1.34-2.15-1.37-0.38-0.58-1.04-1.22-1.06-0.88-1.21-1.31-1.09-1.20-0.41-1.06NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.24-0.42-0.62-1.08-1.40-1.06-1.02-1.36-0.88-0.98-1.22-1.24-1.07-1.28-0.24-0.17F-0.76-1.45-0.96-1.48-1.28-0.35NaN-0.840.43-0.92-1.17-1.37-1.96-1.09-0.92-1.82-1.13-1.46-2.05-0.26-1.38-1.64-1.25-1.31-0.41-1.03-1.31-1.56-0.67-1.17-0.39NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.05-1.07-0.72-1.07-0.01-1.23-0.92-0.95-0.70-0.96-0.30NaN0.00NaNNaN-0.21WNaN-1.09NaN-0.96-1.37-0.77NaN-0.28NaNNaNNaNNaNNaN-0.96-1.17-1.62-1.28-1.29-0.83-0.45-1.24-2.16-1.17-2.20-1.24-0.96-1.16-1.28-0.87-1.31-1.22NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.05-1.31-0.89-1.21-0.09-1.22NaN-1.19NaN-0.77-1.24-0.93NaNNaN-0.82NaNY-1.41-1.28-0.12-0.14-0.36-0.95NaN-0.780.03-0.39-0.20-1.62-1.27-1.41-1.73-1.08-1.28-1.18-0.410.00-1.05-1.24-0.83-1.14-0.99-0.19-0.88-1.30-0.60-1.67-1.22NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.32-1.08-0.57-0.910.34-1.58-1.11 -1.16-0.32-1.28-1.31-1.24-0.22-0.82-1.170.00P-1.12-1.04-0.56-1.23-1.14-1.54-0.79-0.38-0.38-0.02-1.30-0.82-1.17-0.83-1.60-0.98-1.24-1.09-1.26-1.190.38-1.160.00-0.89-1.24-0.69-0.810.26-0.33-0.98-1.19NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.31-1.26-0.84-1.18-0.280.00-1.29-1.17-1.11 -0.58-1.48-1.87-1.640.00-0.95-0.97START M0.00-0.82-0.32NaN-1.07-0.50NaN-0.850.58-0.31NaNNaN-1.52NaN-1.75-0.82-0.34-0.50-0.91-0.99-0.370.00-0.97-0.92-0.79-1.21-1.84-0.54-0.41-0.77-1.12NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN0.00-0.33-0.45-1.140.03-1.82-1.120.00-1.80-0.27NaN-0.12NaNNaN-0.89NaNI-0.11 -0.230.61-0.33-1.370.00-0.050.350.49-0.29-1.14-0.35-0.23-0.78-0.41-0.98-1.19-0.78-0.54-1.00-0.88-0.09-0.85-0.94-0.12-0.82-0.52-0.64-0.35-1.28-0.32NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.23-1.09-0.42-1.31-0.05-1.06-1.25-0.22-1.32-0.95-0.27-0.08-0.19-1.33-1.38-0.80L-0.25-2.500.05-1.14-1.13-0.44NaN-0.360.24-0.29-1.04-1.17-0.33-1.58-0.49-1.18-0.94-0.64-1.30-1.08-0.87-0.48-0.38-0.710.00-0.44-0.96-0.84-0.36-1.33-1.11 NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.39-1.26-0.38-1.200.00-0.52-1.190.33-1.270.00-0.74-0.38-0.20-0.25-1.31-1.19V-0.20-1.310.64-0.20-0.49-0.21NaN0.30-0.55-0.50-0.44-0.730.00-0.270.00-0.300.000.00-1.21-1.04-0.44-0.38-1.01-0.84-0.51-1.22-1.07-0.27-0.34-0.430.00NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.40-1.07-0.37-0.94-0.38-0.80-0.66-0.24-0.34-0.940.000.00-0.31-1.21-0.22-1.41A-1.09-0.99-0.260.55-0.70-1.23-0.730.550.210.53-0.96-0.21-0.26-0.49-0.480.00-0.48-0.50-1.50-1.220.04-1.23-0.87-0.89-0.77-1.22-0.550.000.00-0.44-0.54NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.02-0.99-0.14-0.44-0.86-0.65-0.90-1.11 -1.05-1.28-0.25-0.32-1.26-0.40-0.82NaNG-0.99-0.20NaN0.00-0.42-1.06-0.120.300.33-0.26-0.45-0.63-0.640.00-0.72-0.79-0.73-0.31-1.41-1.25-0.58-1.21-1.21-0.59-1.12-1.06-0.55-0.16-0.28-0.35-0.51NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.09-1.29-0.540.00-1.10-1.020.00-1.85-0.33-1.09-0.54-0.42-1.59-1.19-0.24NaNC-0.77-0.89-1.030.09-0.32-1.29-0.210.480.65-1.40-1.080.05-0.66-0.21-0.88-0.23-1.02-0.85-1.26-0.35-0.70-1.84-1.19-0.480.14-0.85-0.29-0.10-0.42-0.86-0.75NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.45-1.15-0.53-0.55-0.80-0.94-0.44-0.63-1.47-0.93-0.36-0.71-0.32-1.41-1.03-0.11 S-1.27-0.350.11 0.41-1.12-0.800.000.000.00-0.27-1.07-0.12-1.26-0.34-1.18-0.56-1.16-1.20-0.63-0.890.07-1.26-0.47-0.46-0.42-1.03-0.080.16-0.13-0.95-1.08NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.26-0.93-0.23-0.83-0.99-0.33-0.72-1.05-1.20-1.10-1.01-0.78-0.21-0.41-1.23-0.50T-0.31-0.710.56-0.10-1.13-0.48-0.230.520.590.02-1.430.00-1.15-1.11 -1.15-0.41-1.37-0.92-0.98-1.39-0.01-0.62-0.65-0.74-0.36-0.870.00-0.02-0.24-1.28-1.07NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.61-0.93-0.28-1.17-0.85-0.66-1.17-0.36-1.43-1.22-1.38-1.72-0.81-0.19-0.92-1.02NNaN-1.020.00-0.29-0.37-0.49-0.040.530.560.00-0.38-0.23-1.21-0.94-1.38-1.38-1.13-1.470.00-0.73-0.44-1.00-2.02-0.75-0.82-0.56-0.08-0.30-0.45-0.80-1.26NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.34-0.52-0.34-1.17-1.16-1.25-1.11 -1.01-0.54-1.22-1.16-0.92-1.09-0.96-0.96-0.16Q-1.18-0.560.66-0.28-1.52-1.20NaN-1.620.450.13-1.25-0.66-0.84-0.89-0.82-1.52-1.69-1.65-0.90-1.28-0.41-1.16-0.43-0.83-1.13-0.41-0.770.14-0.31-0.19-1.29NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.33-0.820.00-1.19-0.52-0.59-1.64-0.89-1.38-0.45-0.96-1.01-1.75-0.89-0.38-0.99D-1.37-0.89-0.25-0.330.00-1.12NaN-0.140.600.030.00-1.45-1.09-0.41-1.01-0.62-0.92-0.79-0.47-0.79-0.83-0.94-1.43-0.97-1.28-0.39-0.57-0.30-0.19-0.21-0.59NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.25-1.37-0.56-0.63-0.96-1.28-0.53-1.040.00-0.95-0.25-1.52-1.16-1.03-0.23-0.43E-2.17-1.81-0.95-0.77-0.34-1.20-0.89-0.46-0.12-0.04-0.53-0.88-0.27-1.90-0.48-1.48-0.33-0.46-1.74-1.25-0.25-1.22-1.15-1.05-1.23-1.52-1.03-0.45-0.150.00-1.38NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.10-0.29-0.30-1.04-0.96-1.42-1.23-1.31-0.07-1.12-0.71-0.14-1.14-0.890.00-1.52H-0.91-1.000.00-1.47-0.99-1.15-0.67-1.190.490.04-0.50-0.93-1.54-2.06-1.12-1.07-1.00-0.98-0.85-0.49-0.58-1.50-1.08-0.46-1.060.00-0.68-0.29-0.42-0.72-1.37NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.11 -1.14-0.23-1.13-0.61-1.00-0.99-1.94-0.79-1.43-1.72-1.00-1.19-0.17-1.11 -0.23K-0.42-0.25-0.42-1.03-1.14-1.42-0.480.64-0.33-0.04-1.16-0.83-1.12NaN-1.28-0.97-1.13-1.13-0.52-1.010.00-0.65-1.09-0.70-1.16-0.72-0.620.82-0.27-0.34-1.81NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.580.00-0.07-1.09-1.35-0.95-1.35-0.47-1.03-1.28-0.88-1.14-1.03NaN-0.30NaNR-0.620.00-0.23-0.53-1.30-1.27-0.05-0.49-0.67-0.17-1.40-0.89-1.42-0.54-1.21-1.03-1.20-1.15-1.32-1.45-0.28-1.07-0.820.00-1.22-0.30-0.640.74-0.30-1.17-1.28NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.96-0.45-0.13-0.95-0.91-0.90-0.96-0.48-1.29-0.80-0.87-0.98-1.49-0.40-1.67-0.81AmiE I38V 616263646566676869707172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110 111112 113 114 115 116 117 118 119 120Mutation SLQGIMYDPAEMMETAVAIPGEETEIFSRACRKANVWGVFSLTGERHEEHPRKAPYNTLVSTOP *-0.89-0.78-0.39NaN-1.18-1.20-0.31-1.34-1.03-0.88-0.36-1.43-1.07-0.53-0.92-1.35-1.04-1.16-0.98-1.00-1.73-0.65-0.68-1.18-0.31-2.18NaN-1.65-1.21-2.34-0.31-0.69-0.58-2.05-1.95-2.05-0.38-1.65-1.34-1.89-2.79-2.57-2.08-3.00-0.82NaN-1.63-0.41-0.32-1.73-4.14-1.05-0.55-1.53-1.33-0.39-3.35-2.40-1.61-2.76F-0.27-0.11 -1.15-1.52-0.40-1.16-0.18-1.15-0.19-0.41-0.38-1.94-0.48-1.39-0.57-1.16-1.14-0.87-0.29-1.04-1.25-1.05-1.65-0.96-0.93-0.450.00NaN-0.76-1.21-0.26-1.57-0.77-1.97NaN-0.79-1.10-1.70NaN0.00-0.40-0.37-1.62-2.24-1.55NaN-2.05-1.94-1.45-1.77-2.54-0.42-2.31-1.92NaN-0.20NaN-2.11 -0.52-0.49W-0.89-0.59-1.33NaN-1.09NaNNaN-1.66-0.06-0.46-1.04-1.07-0.89-1.41-1.26-2.42NaN-0.92-1.00NaN-1.02-1.51-0.70-2.01-0.66-1.95NaNNaN-1.09NaN-0.14-1.09-1.49NaNNaNNaN0.00-2.02-1.09-0.72-1.53-1.51-2.40-1.94-2.90NaNNaN-1.87NaN-1.79NaN-1.02-1.02NaNNaNNaNNaNNaN-0.83NaNY-0.19-1.14-2.06-1.11 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-1.98-1.56NaNNaN-1.47-2.37NaNNaN-1.92-0.57-0.380.01NaNNaNNaNNaN0.04NaNI-1.03-0.48-0.87-0.790.00-0.20-0.85-1.29-0.490.06-0.61-0.15-0.14-0.81-0.50-1.21-0.52-0.420.00-1.37-1.42-0.42-1.14-0.85-0.740.00-0.24-0.40-0.39-1.37NaN-0.26-0.41-1.80-0.74-0.68-1.82-2.37-1.52-0.28-1.500.54-0.62-1.87-1.68NaN-1.73-2.08NaN-1.67-2.22-0.63-1.46-2.00-1.31-2.57-0.49-0.51-1.120.48L-1.280.00-0.73-0.98-0.63-0.37-0.65-1.04-0.27-0.12-0.210.040.04-0.590.12-1.16-0.45-0.28-0.98-0.52-1.11 -0.56-1.14-0.98-0.62-0.48-0.09NaN-0.33NaN-1.79-0.93-0.45-2.10-2.04-1.12-0.45-1.63-0.36-0.29-1.820.00-1.78-2.02-1.78-0.57-0.80-2.71-0.89-0.74-0.56-0.34-1.67-1.37-0.41-2.14-1.95-2.11 0.00-0.34V-0.84-0.99-1.51-0.290.24-0.27-1.07-0.53-0.350.04-0.32-0.25-0.35-0.43-0.82-0.300.00-0.220.44-1.40-0.68-0.09-0.74-0.84-0.24-0.28-0.35NaN-0.42-0.29-2.24-0.35-0.54-0.57-1.600.00-2.40-0.360.00-0.63-1.95-0.12-1.77-0.60-0.71NaN-1.81-0.63-0.30-2.72-1.82-0.71-1.74-0.29NaNNaN-2.25NaN0.160.00A-0.40-1.58-1.08-0.76-0.58-0.63-1.16-0.83-0.330.00-0.29-0.79-1.070.24-0.450.00-0.340.00-0.47-0.59-1.020.19-0.96-0.66-0.06-1.48-2.45-0.090.470.00-1.970.020.170.00-0.83-0.57-1.89-0.64-0.39-1.98-0.68-0.12-0.74-1.04-1.36NaN-2.19-1.30-0.63-1.52-0.80-0.30-1.280.00-0.59-1.31-1.81-0.270.72-0.32G-1.03-1.19-0.850.00-1.15-1.02-1.37-0.54-0.90-0.25-0.35-1.03-1.24-0.24-1.11 -0.44-0.27-0.40-0.69-1.030.00-0.39-0.48-1.77-0.08-1.60-0.84-0.11 0.33-0.59-0.64-0.46-0.53-0.77-0.37-0.40-0.330.00-0.76NaN-1.34-2.18-1.790.00-0.61-0.48-1.82-0.42-0.20-3.20NaN-0.56-2.71-0.55-1.21-1.31NaN-2.73-0.56-0.50C-0.24-0.86-1.39-0.17-0.58-0.62-0.13-0.88-0.49-0.33-0.93-0.99-0.88-0.650.12-0.78-0.89-0.180.73-1.12-0.42-0.57-1.01-1.05-0.80-0.59-0.07-0.22-0.09-1.530.00-0.65-0.39-0.55-0.85-0.02-0.34-0.29-1.59-0.29-0.690.11 -2.10-0.50-1.82-0.15-2.06-5.47-1.06-1.60-1.98-0.33-1.11 -0.12NaN-0.09-1.89NaN0.35-1.23S0.00-0.39-1.07-0.52-0.93-0.64-0.44-0.58-0.38-0.21-0.71-0.97-1.20-0.19-0.43-0.54-1.360.10-0.40-0.47-0.78-0.51-1.08-0.78-0.48-1.41-0.250.000.14-0.20-0.61-0.46-0.13-0.42-0.39-1.27-1.12-0.67-1.86-0.490.00-2.22-0.86-0.41-1.82-0.28-1.85-1.47-1.27-2.24-0.69-0.70-1.73-0.36-0.33-0.88-0.42-0.300.42-3.23T-0.230.41-0.91-1.07-0.71-0.32-0.67-0.58-0.20-0.04-0.57-0.60-0.50-0.510.00-0.50-1.150.06-0.74-0.43-1.21-0.38-1.070.00-0.62-0.49NaN-0.590.00-0.34-1.68-0.700.02-0.39-0.48-0.13-2.16-2.10-1.65-1.49-0.65-2.230.00-2.15-1.83NaN-1.54-2.28-1.65-1.78-0.52-0.64-0.83-0.34-0.35NaN-0.960.00-0.29-2.33N-0.79-1.05-1.49-1.05-0.63-1.06-0.28-0.26-0.50-0.11 -0.78-1.05-1.24-0.07-0.95-1.17-0.70-0.51-0.74-0.96-1.09-0.48-1.30-0.95-0.19-0.52NaN-0.310.30-1.72NaN-1.01-0.24-1.000.00-1.57-1.60-2.20NaN-1.66-1.78NaN-0.87-1.35-2.11 NaN-0.46-1.95-1.40-0.38-1.61-0.05-0.53-0.27NaN-0.320.00-0.60-1.51-1.63Q-1.33-1.740.00-1.66-0.98-0.31-0.82-0.97-0.170.18-0.23-0.79-1.09-0.11 -1.09-0.99-1.67-0.07-1.32-0.51-1.190.10-0.94-1.10-0.18-0.81-1.23-2.280.25-1.17-1.87-0.170.26-1.18-0.26-2.76-2.50-1.75NaN-2.62-1.28-0.34-1.69-3.98-1.02-0.52-0.38-0.95-0.44-0.22-4.96-0.57-1.56-0.18-0.05-1.29-2.03-1.23-0.16-1.65D-0.93-2.42-1.04-0.29-1.03-1.14-0.200.00-0.80-0.12-0.32-1.18-0.99-0.14-1.00-0.25-2.01-0.67-1.10-1.91-0.580.01-0.65-1.37-0.05-1.65-0.83-2.080.57-0.33-2.43-1.660.41-2.15-0.20-0.30-1.82-0.26-1.29-1.77-2.25-2.11 -1.78-0.18-0.67-1.49-0.73-0.50-0.01-0.69-1.63-0.22-1.48-1.50-0.78-0.85-0.22NaN-0.95-0.19E-1.09-1.28-0.54-1.07-0.97-0.93NaN-0.14-0.480.060.00-1.40-1.330.00-1.26-0.95-0.19-0.02-1.13-2.60-1.11 0.000.00-1.470.00-0.69NaN-1.530.73-1.17-1.38-0.640.32-0.51-1.04-2.50-1.66-1.74-0.17NaN-1.46-1.52-2.11 -1.620.00NaN-1.620.000.00-1.39-2.25-0.86-0.20-0.15-1.09-1.49NaN-2.25-1.68-1.89H-0.96-1.13-0.39-1.40-1.24-0.59-0.23-0.680.030.00-0.69-1.17-1.03-0.60-0.79-1.30-0.87-0.54-1.11 -1.37-0.92-0.04-0.86-1.33-0.45-1.12NaN-1.43-0.11 -2.00-1.56-0.86-0.32-1.84-0.40-2.89NaN-1.63-2.45-1.31-2.00-1.89-1.62-1.95-2.09-0.120.00-2.41-0.770.00-0.240.01-2.11 -0.68NaN-0.20-0.73-2.57NaN-3.41K-0.86-0.96-0.68-1.16-0.99-0.26-0.57-0.80-0.030.39-0.22-0.48-0.26-0.10-0.28-0.98-1.14-0.05-1.22-1.09-1.540.02-0.63-0.52-0.29-1.45NaN-1.310.32-1.95-1.55-0.130.00-1.31-0.22-1.46-2.45NaNNaNNaN-1.54-1.81-1.67-1.87-0.51-0.37-3.08-0.49-0.27-1.25-1.820.020.00-0.53NaN-1.26-0.42-1.42-1.18-1.21R-1.87-1.28-0.59-0.84-1.12-0.65-0.67-1.040.11 0.21-0.58-0.07-0.77-0.12-0.46-1.14-1.40-0.17-1.17-0.70-1.040.07-1.23-1.06-0.29-1.63-1.42-0.200.00-1.86-0.350.00-0.23-1.61-0.75-2.98-0.52-1.33-1.57-2.09-1.76-0.80-1.96-1.26-2.120.00-0.51-2.13-3.68-0.51-1.010.00-0.39-0.15-0.56-1.22-4.41-1.59-0.36-1.81AmiE I38V 121122123124125126127128129130131132133134135136137138139140141142143144145146147148149150151152153154155156157158159160161162163164165166167168169170171172173174175176177178179180Mutation LIDNNGEIVQKYRKIIPWCPIEGWYPGGQTYVSEGPKGMKISLIICDDGNYPEIWRDCAMSTOP 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-1.60-0.68-2.50-1.84-0.37-1.46NaN-0.48-2.14-2.05-0.53-3.29-0.57-0.22NaN-1.76-0.56-3.08-1.57-1.52-1.49-0.50NaN-0.15NaN-0.19-0.40-1.47-0.20-2.51-2.33F-0.30-0.45-1.95-1.82-0.52-1.09-0.41-0.44NaN-1.91NaN-0.27-1.65-1.82-0.370.66-1.30-0.24-0.53NaN-0.22-1.51-0.46NaN-0.54-1.55-1.63-1.68-1.82-2.69-0.08-0.84-0.65-1.89-1.68-0.95-1.79NaN-1.18NaN-0.40-1.96-0.48-0.30-0.06-0.40NaNNaN-1.43-1.49-0.27-2.15NaN-0.27NaN-1.80-1.81-0.16-0.98-1.13W-0.24-1.26NaN-1.19NaNNaN-1.15NaN-1.09-2.43-2.14NaNNaNNaN-1.36-1.23-1.980.00-0.49NaN-1.36-1.75-0.580.00-1.51NaN-1.74NaNNaN-1.77-2.40-1.78-1.74NaN-1.53NaNNaNNaNNaNNaNNaN-1.23-1.12NaN-0.30-0.35-1.92NaN-1.40-0.87NaN-1.72NaNNaN0.00-2.07NaN0.07-1.55NaNY-1.92-1.80-0.42-1.17-0.21NaN-0.49-1.37NaN-1.98-1.620.00-2.22-1.82-1.60-0.35-3.35-0.68-0.25NaN-0.68-1.74-0.42NaN0.00-1.81-1.61NaN-0.76-1.820.00-2.51-0.48-1.54-1.65NaN-1.43NaN-1.49-1.59-1.44-5.39-2.44-1.52-0.86-0.43-0.37-0.28-1.64-0.270.00-2.77NaN-1.84-1.26-2.89-0.270.00-1.46-0.99P-1.57-1.93-1.97-0.20NaN-1.26-1.91-1.72-2.05-1.48-2.13-1.32-1.78-1.59-2.46-1.700.00-1.86-1.850.00-1.66-1.750.12NaN-2.200.00-1.75-1.35-0.97-1.60-1.95-1.95-0.81-0.50-1.710.00-1.26-1.61-1.20-2.43-1.60-0.74-1.56-1.62-1.18-2.09-2.66-1.69-1.86-2.82-3.980.00-2.20-1.55-1.34-1.65-2.04-2.12-0.84-2.93START M-0.38-0.45NaN-1.67-0.05NaN-0.24-0.37-0.99-1.67-0.79NaN-1.06-0.22-0.170.54-2.11 -2.62-1.89-0.87-0.08-2.68-0.07NaN-1.65NaNNaN-0.520.40-1.22-2.62-3.48-3.26-0.78-1.97NaN-1.49NaN0.00-0.35-0.20-1.45-0.22-0.10-0.39NaN-1.75NaN-1.94NaN-1.21-1.19NaN-0.08NaNNaNNaN-0.76-1.780.00I-1.570.00-2.33-0.77-0.32NaN-0.340.00-0.26-1.39-1.67-1.89-0.46-2.620.000.00-5.47-1.71NaNNaN0.00-1.63-0.59NaN-1.85-1.09-1.24-1.23NaN-0.53-1.63-0.65-1.82-0.24-1.73-1.72-0.57-2.28-0.09-1.410.00-1.33-0.420.000.00-2.12-1.94-1.70-2.15-0.26-2.32-3.27-1.250.00NaN-0.65-2.23-1.04-1.73-0.17L0.00-0.23-2.36-2.60-0.48-1.23-0.37-0.83-0.52-1.09-2.46-1.90-2.06-2.25-0.190.43-0.63-1.07-1.74-0.19-0.49-1.79-0.54-0.25-2.30-0.49-2.20-0.99-0.55-1.76-1.91-1.54-1.85-0.60-2.06-0.24-1.76-1.40-0.32-0.90-0.60-0.630.00-1.22-0.49-1.46NaN-3.11 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-0.53NaN-1.76-0.390.00-0.20-1.46-1.740.000.00-0.81-1.96-1.53-0.82-2.05-0.250.00NaN-2.680.00-1.49-1.61-1.91-2.10-2.06-1.65-1.95-0.74-0.26-0.130.00-2.22-1.93NaN-0.20-1.25-0.37-0.33-0.23-0.18-0.41-1.04C-2.22-1.71-1.86-0.60-0.10-0.14-0.24-1.45-0.81-1.50-2.21-0.08NaN-2.90-1.82-0.11 -1.53-0.070.00-1.45-1.08-1.850.31-0.12-0.27-1.79-0.35-0.21-1.15NaN-0.41-0.55-0.36-0.57-0.65NaNNaN-0.27NaN-1.86-1.35-1.72-0.89-1.980.960.00-1.67-0.81-0.35-0.95-0.22-1.88-1.55-2.04-0.09-1.89NaN0.00-1.17-1.98S-0.28-1.45-0.48-0.10-0.26-0.090.03-1.10-1.62-2.18-1.77-0.81-0.61-1.62-1.12-0.07-0.65-1.46-0.56-0.15-0.40-1.680.07-0.30-0.71-0.48-0.820.07-0.11 -0.76-1.24-1.950.00-0.35-1.25-0.31-1.56-0.34-1.32-2.60-0.860.00-0.48-0.53-0.09-0.64-1.27-3.58-0.45-0.28-1.02-0.65-3.95-0.71-0.73-0.48-2.36-0.29-0.46-1.16T-1.35-0.96-2.45-0.84-0.23-1.21-0.04-0.53-1.11 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-0.27NaN-2.40NaNNaN-1.39-1.49NaN-1.620.000.00-0.45-0.22-0.89-1.85-0.34-1.87NaN-1.720.00NaN-0.16-1.19E-2.14-2.22-0.40-1.05-0.26NaN0.00-1.26-0.39-0.89-0.46NaN-1.52-0.18-3.79-1.46-2.43-1.68-1.57NaNNaN0.00-0.71NaN-2.06NaN-1.96-0.09-0.15-2.49-1.17-2.11 -1.910.00-1.94-1.26-0.22-1.18NaN-0.30-1.26-1.85-2.99-1.33-1.12-1.76-0.05-0.18-1.23-1.57-1.67-2.380.00-2.04-0.62-1.68-0.24-1.32-1.27-1.39H-2.11 -1.42-0.64-0.530.04-1.02-0.03-1.73-1.49-0.86-1.46-0.19-1.61-1.74-1.82-0.28-2.20-1.44-1.52NaN-0.45-1.55-0.09NaN-0.40-1.48-5.09-0.06-0.41-1.83-0.42-2.12-1.79-0.43-2.17-1.67-2.57NaN-1.72-2.20-1.65-2.00-1.46-2.40-2.54-1.68-0.63-0.62-1.89-0.89-0.33-2.04-2.01NaNNaN-1.83-0.62NaN-2.25-1.75K-2.73-2.78-2.88-0.770.38NaN-0.05-1.08-0.34-0.940.00NaN-0.400.00-5.31-2.24-2.68-1.34-3.29NaN-0.76-0.46-0.13-1.02-2.82NaN-3.380.26-0.09-1.10-1.91-2.05-1.82-0.20-1.76NaN0.00NaN-0.080.00NaN-6.23-1.30-1.52-1.21-1.98-2.45-2.17-2.96-0.16-2.09-1.08-0.36-2.85-2.01-0.65NaNNaN-2.33-0.40R-1.64-1.78-1.32-0.800.10-0.630.02-2.730.04-0.69-0.49NaN0.00-0.47-1.94-1.84-1.17-0.70-0.60-0.51-0.96-1.530.05-0.20-1.63-0.77-1.13-0.11 0.06-1.44-1.73-1.76-1.82-0.57-1.44-0.82-0.32-0.80-0.36-0.31-2.42-2.29-3.59-1.59-1.02-0.55-2.15NaN-1.11 -1.55-1.75-1.39-1.76-1.65-0.350.00-2.12-0.29-1.83-0.62AmiE I38V 181182183184185186187188189190191192193194195196197198199200201202203204205206207208209210211 212213214215216217218219220221222223224225226227228229230231232233234235236237238239240Mutation KGAELIVRCQGYMYPAKDQQVMMAKAMAWANNCYVAVANAAGFDGVYSYFGHSAIIGFDGSTOP *-0.65-1.99-2.11 -0.31-1.10-1.38-1.96-2.17-0.50-0.08NaN-0.55-2.48-0.22NaN-1.60-0.36-2.14-0.28-0.74-2.19-1.37-3.36NaN-0.53-1.17NaN-1.28-0.16-1.76-1.34-3.63-0.48-0.45-1.40-1.65-1.26-1.76NaNNaN-1.68NaN-1.74-3.18-1.78-1.93-0.12-2.28-0.25-2.00NaN-2.44-1.82-1.74-2.09-2.11 -5.18-1.68-1.03-3.48F-1.91-7.63-1.71-1.65-0.40-0.16-0.44-1.44-0.34-1.09NaN-0.25NaN-0.39NaN-1.20-1.14-1.22NaN-1.51-0.18-1.20-1.53-2.12NaNNaN-0.99-1.40-0.64-1.24-3.98-1.76-0.41-0.71-0.43-0.98-1.26NaNNaN-0.67-1.68-2.740.00-1.32-1.58-0.09-0.08-1.190.320.00NaN-2.25-2.85-1.38-0.74-0.37NaN0.00-1.44-1.30W-2.37-1.88-2.46NaN-0.73NaNNaN-0.84-0.62NaNNaN-2.38-1.48-1.04NaN-1.11 NaN-1.67NaN-1.76-0.56-0.74NaN-1.09NaNNaNNaNNaN0.00-1.21-1.95NaN-0.93-1.48-1.21-1.32NaNNaNNaNNaNNaN-1.23-0.84NaNNaNNaNNaNNaNNaN-0.30NaN-1.72-1.04-0.99-1.65-1.60-1.87NaNNaNNaNY-2.41-1.76-3.70NaN-2.41-1.90-1.87-1.46-0.310.13NaN0.00-2.930.00-0.90-1.53NaN-0.46-1.85-1.75-0.93-1.17-1.55-1.47-2.36NaN-1.34-0.87-0.85-2.20-0.40-0.79-0.280.00-3.54NaN-1.49NaN-0.35NaN-2.25-1.55-0.25-0.28NaN-0.590.00-0.760.000.23NaN-0.18-1.48-1.51-1.730.64-1.58-0.68-0.32-2.15P-2.12-1.74-1.51-2.14-1.83-1.95-1.52-0.75-1.30-0.07-3.02-1.830.53-1.540.00-1.03-1.53-1.18-0.87-0.87-1.39-1.65-1.59-0.92-2.01-0.93-1.66-0.46-1.63-1.41-1.77-1.93-1.44-1.76-2.61-0.86-2.09-0.73-2.66-0.61-0.94-1.80-2.00-2.16-2.23-2.02NaN-1.51-1.62-1.60NaN-0.56-2.02-1.54-1.75-2.12-1.93-2.25-1.73-1.95START M-0.53-2.27-1.13NaN-1.25-0.40-1.50NaN-1.27NaNNaN-1.280.00-1.32NaN-1.32-0.36-1.13-0.43-2.671.040.000.00NaN-0.20-0.820.00NaNNaN-1.00NaN0.46NaN-1.09NaNNaN-0.17NaN-1.04NaNNaN-2.08-1.43-1.52NaN-0.91NaNNaNNaN-0.56NaNNaNNaN0.88-0.56-0.64NaNNaN-1.19NaNI-1.78-3.05-1.88NaN-1.630.00-0.30NaN-2.29-0.91-1.64-1.96-0.25-2.00-2.15-1.50-0.29-1.35-0.25-2.030.71-0.27-0.21-1.80-1.25-1.19-0.20-1.10NaN-2.31-0.35-0.28NaN-1.49-0.36-1.72-0.44-1.98-0.54-0.95-1.63-1.77-1.42-1.87NaN-0.22-1.22-0.36-1.06-0.61NaN-1.40-0.88-0.670.000.00-1.47-1.02-1.98-1.87L-1.58-2.20-2.61-1.960.00-1.14-1.05-0.52-2.89-0.18-3.73-1.90-0.50-1.55-0.33-2.32-1.13-1.31-0.57-1.150.85-0.08-0.49-1.85-1.52-2.07-0.59NaN-0.35-2.35-2.160.03-3.51-4.37-1.11 -2.58-0.59-2.35-2.27-1.04-1.66-2.60-0.65-1.44NaN-0.57-2.27-2.16-1.65-0.27-1.34-0.58-2.54-0.64-1.03-0.98-1.77-0.53-1.91-1.71V-1.86-0.76-0.41-0.26-1.10-0.310.00NaN-1.70-1.48-0.30-2.11 -0.40-3.10-1.32-0.20-0.57-0.47-0.67-1.820.00-0.54-0.28-0.33-2.12-0.06-0.48-0.35-2.20-0.34-1.740.190.52NaN0.00-0.360.00-0.18NaN-0.30-0.36-1.08-0.73-0.20-0.140.00NaN-0.65-1.55-0.93-0.18-1.57-1.94-0.50-0.71-0.65-0.89-1.05-0.14-0.40A-1.83-1.230.00-1.03-2.61-1.15-0.53-2.02-0.390.39-0.52-1.44-0.43-0.15-0.950.00-0.42-0.90-0.40-1.21-0.37-0.11 -2.040.00-2.800.00-1.700.00-2.990.00-1.92-1.08-2.16-2.20-0.550.00-0.540.00-1.300.000.00-1.57-1.48-0.93-0.74-0.37-1.330.15-1.88-1.84-0.38-1.32-3.090.00-1.79-1.96-0.91-2.12-0.50-0.77G-1.660.00-0.94-0.30-1.99-1.46-0.86-0.56-0.79-0.320.00-3.46-0.50-2.23-1.81-0.32-2.23-0.51-2.67-1.56-0.85-1.12-1.26-0.44-1.40-0.46-2.57-0.35-0.70-0.73-1.74-0.75-0.41-2.11 -0.89-0.80-1.23-0.65NaN-0.03-0.750.00-2.24-0.270.00-0.12NaN-0.20NaN-1.180.00-0.61-0.64-0.26-1.28-4.060.00-1.48-0.220.00C-2.27-0.35-1.77-1.24-1.48-1.73-0.50-0.320.00NaN-0.39-0.48-0.45-0.33NaN0.020.30-1.22-0.75-1.96-0.51-0.50-1.68-0.08-1.900.10-1.93NaN-0.36-0.44-2.210.510.00-0.41-1.68-0.90-0.53NaN-1.68NaNNaN-0.61-0.67-1.10-0.13-0.55-0.11 -0.26-0.100.25-0.12-0.83-0.530.77-2.31-1.32-0.41-1.06-0.74-0.21S-1.84-1.19-0.72-5.04-0.75-0.65-1.57-0.61-0.310.84-0.46-1.22-0.47-0.08-0.44-0.22-1.07-1.15-0.99-1.04-0.56-0.91-2.770.00-2.00-0.35-2.76-0.35-0.93-0.93-0.64-0.40-0.57-1.88-1.87-0.56-1.90-0.43-0.40-0.21-0.14-1.31-0.53-3.20-0.28-1.09-0.690.00-0.66-0.21-0.23-1.300.00-0.50-1.25-0.970.10-0.83-2.75-0.40T-1.27-1.80-0.65-1.93-1.94-0.55-1.41-1.48-1.770.37NaN-1.74-0.39-1.15-0.14-0.52-1.04-1.09-0.19-1.46-0.19-0.23-0.47-0.47-0.98-0.41-0.52-0.29-2.41-0.78-1.140.40-0.80-1.46-2.06-0.50-0.92-0.51-0.58-0.300.41-1.43-0.97-2.42-1.23-0.29-1.44-0.38-1.48-0.70NaN-1.28-1.20-0.36-1.02-0.86-1.48-1.97NaN-2.45N-0.61-1.44NaN-2.31-2.35-0.27-1.68-1.52-1.58-0.54-1.90-1.06-0.95-0.29-0.84-0.42-0.35-0.41-1.35-0.750.250.00-1.090.26-0.35-2.07-1.46-1.93NaNNaN0.000.00-2.09-1.13-3.18NaN-2.31-1.680.00-1.36-1.98-1.31-0.41-0.39-1.26-1.81-0.46-0.31-0.17-1.31NaN-0.24-0.63-1.80-0.77-0.42-0.77-1.97-0.18NaNQ-0.71-2.03-1.83-0.42-2.14-1.49-1.29NaN-2.540.00-1.40-1.55-0.78-0.45NaN-0.95-0.50-0.800.000.00-0.17-0.83NaN-1.52-0.85-1.85-1.02-1.14-2.15-1.41-1.430.76-2.25-2.99-3.10NaN-0.30-1.93NaNNaN-2.83-1.86-1.85-1.72NaN-0.78-1.36-1.13-0.98NaNNaN-0.32-1.43-0.28-2.95-1.78-2.02NaN-1.84-1.73D-2.02-0.68-1.60-0.33-6.50-2.16-0.53-1.07-2.12-0.47-0.51-1.02NaN-0.88NaN-0.43-1.360.00-2.28-1.91-0.78-1.47-1.72-0.29-1.40-1.79NaN-0.37-1.68-1.79-0.41-0.34-1.81-1.58-0.45-0.44-2.31-2.44-0.30-0.19NaN-0.87-2.040.00-0.41-0.59-0.24-1.36-0.09NaN-0.29-0.67-2.61-0.71-2.23-1.54-0.42-1.360.00-0.52E-0.67-1.53-0.520.00-1.80NaN-1.95NaN-3.02-0.09-0.80-2.00NaN-1.26NaN-1.41-0.11 0.25-0.63-0.86-1.58-1.51NaN-2.12-0.17-0.30NaNNaNNaN-0.47-1.28-0.75NaN-1.32-1.81-1.58-0.72-0.28NaN-1.32-0.26-4.44-1.63-0.24-0.93-1.44-1.52-1.64NaN-1.95NaN-1.28-2.35-1.61-2.49NaN-1.35-1.08-0.17-1.98H-1.92-2.36-3.02-1.42-2.46-1.79-1.63-0.27-1.61-0.26-1.05-0.51-1.33-0.41-0.19-1.98NaN-0.52-0.32-0.79-0.35-1.32-1.36-1.88-1.38-3.07-2.80-1.40-1.30-1.62-1.42-0.16-2.57-0.62-1.76NaN-2.07-2.62-0.79NaN-1.81-1.48-0.24-0.79-1.52-0.67-0.08-0.05-0.26-0.30NaN0.00-1.94-1.06-2.15-2.22-1.89-0.99-0.50-1.65K0.00-2.97-2.23-0.40-1.44NaN-1.63-1.84NaN-0.04NaN-1.71-0.53-2.25NaN-2.590.00-1.75-0.30-0.80-0.67-0.83-0.25-1.620.00NaN-0.42-1.58-1.00-1.49-0.390.07-1.36-2.41NaN-1.40-2.14-2.41-0.30NaN-2.27-1.19-2.17-1.17-1.01-0.81-1.44-0.30NaNNaN-1.48-2.83-3.03-1.52-2.17-3.37NaN-2.83-0.87-1.79R-0.31-1.47-1.61-4.18-1.87-1.97-1.920.00-0.49-0.14-0.97-2.56-1.20-1.62-0.83-1.65-0.20-1.30-0.33-0.80-0.11 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NaN-0.28NaNNaN0.17-0.14-0.27-0.65-0.01-0.16NaNNaN-0.29-0.59-0.48-0.51-0.65-0.34-0.39-0.62-0.71-0.32-0.31-0.10-0.14-0.660.37-0.46-0.18I-1.86-0.56-1.22-0.54-1.51-0.71-0.27NaN-0.05-0.27NaN-1.62-0.86-0.98-2.93-0.34-0.51-0.56-0.08-0.53-0.020.14-0.18NaN-0.51-0.24-0.24-0.39-0.40-0.33-0.41-0.31-0.96-0.07-0.38-0.08-0.10-0.550.16-0.14-0.28L-0.32-0.51-1.38-0.51-1.47-0.69-0.24-0.17-0.990.08-1.53-1.75-0.91-0.47-1.83-1.35-0.35-0.65-0.62-0.76-0.310.00-1.59-0.45-0.59-0.86-0.14-0.48-0.53-0.34-0.33-0.42-0.39-0.20-0.31-0.160.00-0.270.12-0.06-0.18V-1.52-1.21-0.53-0.58-1.96-0.99-0.57-0.790.00-0.72-0.24-0.41-0.10-0.86-0.23-0.89-0.15-0.810.00-0.36-0.65-0.48-1.63NaN-0.21-0.560.01-0.260.00-0.25-0.24-0.69-0.810.00-0.17-0.05-0.14-0.690.12-0.24-0.21A-0.67-1.68-1.13-1.67-1.36-1.64-0.44-1.28-0.43-0.29-0.790.00-0.38-0.120.00-1.31-0.20-0.15-0.44-0.39-0.05-1.46-0.22-1.140.00-0.26-0.07-0.06-0.060.00-0.04-0.45-0.23-0.07-0.020.050.080.020.19-0.04-0.08G-1.66-1.65-0.36-2.53-1.29-0.87-1.36-0.11 -0.41-0.93-0.26-0.69-0.19-0.79-0.50-0.30-0.31-1.03-0.45-0.31-0.48-2.29-1.28-0.570.02-0.41-0.860.00-0.16-0.08-0.13-0.31-0.44-0.160.000.220.05-0.190.300.050.00C-0.97-0.56-1.19-1.23-0.19-0.27-0.65-0.09-1.79-0.45-1.07-0.30NaN-0.83-0.08-1.89-0.69-0.53-0.54-0.59-0.16-2.050.01-0.30-0.22-0.47-0.37-0.30-0.24-0.28-0.260.00-0.49-0.070.50-0.04-0.13-0.680.13-0.24-0.14S-0.37-0.63-0.91-0.65-0.71-0.68-0.71-0.65-1.35-0.13-2.20-0.36-0.87-0.64-0.58-0.46-0.34-0.43-0.90-1.86-0.43-1.61-0.11 -0.570.00-0.07-0.16-0.200.100.15-0.02-0.37-0.17-0.16-0.160.17-0.06-0.200.20-0.20-0.11 T-0.45-2.02-1.06-1.44-2.27-0.970.00-2.12-1.320.00-1.53-0.61-0.63-0.39-0.57-0.84-0.27-0.20-1.24-0.91-0.34-1.800.00-0.98-0.080.000.00-0.160.060.01-0.13-0.42-0.25-0.03-0.220.13-0.11 -0.200.22-0.22-0.24NNaN-1.44-2.03-1.66-0.35-0.30-0.14-0.85-1.230.04-0.06-2.20-0.27-0.48-2.53-1.76-0.610.00-2.47-0.91-0.28-1.70-0.18NaN-0.01-0.05-1.24-0.21-0.26-0.05-0.09-0.210.25-0.25-0.100.21-0.05-0.340.28-0.07-0.14Q-0.11 -1.72-0.42-2.03-1.55-0.84-0.39-1.23NaN-0.01-0.65-1.65-0.26-0.34-1.31-1.03-0.140.22-2.05-0.32-0.12-0.59-1.45-1.950.06-0.21-0.86-0.11 -0.550.010.00-0.34-0.10-0.22-0.090.250.04-0.220.20-0.15-0.23DNaN-0.92-0.07-1.75-0.70-1.00-0.99-1.23-0.24-1.010.00-0.53-0.11 NaN-2.20-1.41-0.27-0.23NaN-0.53-1.00-1.53NaN-0.95-0.37-0.34-1.17-0.11 -0.23-0.010.05-0.18-0.40-0.010.050.380.25-0.380.580.020.14ENaN-1.330.00-1.36NaN-2.00-0.73NaN-1.18-0.48-0.08-2.020.00-0.22-0.41-1.100.00-0.65-0.330.00-0.96-1.47NaNNaN-0.39-0.27-0.83-0.10-0.560.120.12-0.12-0.12-0.11 0.040.290.18-0.260.350.000.07H-1.02-1.81-1.19-1.91-0.26-0.30-1.07NaN-1.92-0.14-0.51-0.25-0.71-0.89-1.55-1.16-0.64-0.11 -1.50-1.24-0.22-1.57NaN-0.32-0.19-0.21-1.26-0.25-0.67-0.20-0.17-0.36-0.44-0.12-0.260.04-0.08-0.450.16-0.35-0.23KNaN-1.19-0.30NaN-1.76-0.03-1.23-0.77NaN-0.28-0.54-1.30-0.220.00-1.95-0.55-0.13-0.44NaN-0.370.03-1.74NaNNaN0.07-0.20-0.53-0.22-0.640.02-0.07-0.40-0.15-0.35-0.160.19-0.15-0.280.070.150.04R-0.71-1.62-1.80-1.23-1.630.00-1.42-0.19NaN-0.27-1.70-1.86-1.07-0.12-2.520.00-0.38-0.55-1.29-0.830.00-0.72-1.660.00-0.060.06-0.96-0.33-0.800.10-0.32-0.400.01-0.39-0.230.00-0.11 -0.35-0.02-0.05-0.10Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged AmiE I38V 123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960Mutation MRHGDISSSNDTVGVAVVNYKMPRLHTAAEVLDNARKVAEMIVGMKQGLPGMDLVVFPEYSTOP *-1.16-0.16-1.42-1.15-1.09-1.28NaN-0.27-0.09-1.18-1.38-1.12-1.38-0.88-1.40-1.16-1.34-2.15-1.37-0.38-0.58-1.04-1.22-1.06-0.88-1.21-1.31-1.09-1.20-0.41-1.06NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.24-0.42-0.62-1.08-1.40-1.06-1.02-1.36-0.88-0.98-1.22-1.24-1.07-1.28-0.24-0.17F-0.76-1.45-0.96-1.48-1.28-0.35NaN-0.840.43-0.92-1.17-1.37-1.96-1.09-0.92-1.82-1.13-1.46-2.05-0.26-1.38-1.64-1.25-1.31-0.41-1.03-1.31-1.56-0.67-1.17-0.39NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.05-1.07-0.72-1.07-0.01-1.23-0.92-0.95-0.70-0.96-0.30NaN0.00NaNNaN-0.21WNaN-1.09NaN-0.96-1.37-0.77NaN-0.28NaNNaNNaNNaNNaN-0.96-1.17-1.62-1.28-1.29-0.83-0.45-1.24-2.16-1.17-2.20-1.24-0.96-1.16-1.28-0.87-1.31-1.22NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.05-1.31-0.89-1.21-0.09-1.22NaN-1.19NaN-0.77-1.24-0.93NaNNaN-0.82NaNY-1.41-1.28-0.12-0.14-0.36-0.95NaN-0.780.03-0.39-0.20-1.62-1.27-1.41-1.73-1.08-1.28-1.18-0.410.00-1.05-1.24-0.83-1.14-0.99-0.19-0.88-1.30-0.60-1.67-1.22NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.32-1.08-0.57-0.910.34-1.58-1.11 -1.16-0.32-1.28-1.31-1.24-0.22-0.82-1.170.00P-1.12-1.04-0.56-1.23-1.14-1.54-0.79-0.38-0.38-0.02-1.30-0.82-1.17-0.83-1.60-0.98-1.24-1.09-1.26-1.190.38-1.160.00-0.89-1.24-0.69-0.810.26-0.33-0.98-1.19NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.31-1.26-0.84-1.18-0.280.00-1.29-1.17-1.11 -0.58-1.48-1.87-1.640.00-0.95-0.97START M0.00-0.82-0.32NaN-1.07-0.50NaN-0.850.58-0.31NaNNaN-1.52NaN-1.75-0.82-0.34-0.50-0.91-0.99-0.370.00-0.97-0.92-0.79-1.21-1.84-0.54-0.41-0.77-1.12NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN0.00-0.33-0.45-1.140.03-1.82-1.120.00-1.80-0.27NaN-0.12NaNNaN-0.89NaNI-0.11 -0.230.61-0.33-1.370.00-0.050.350.49-0.29-1.14-0.35-0.23-0.78-0.41-0.98-1.19-0.78-0.54-1.00-0.88-0.09-0.85-0.94-0.12-0.82-0.52-0.64-0.35-1.28-0.32NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.23-1.09-0.42-1.31-0.05-1.06-1.25-0.22-1.32-0.95-0.27-0.08-0.19-1.33-1.38-0.80L-0.25-2.500.05-1.14-1.13-0.44NaN-0.360.24-0.29-1.04-1.17-0.33-1.58-0.49-1.18-0.94-0.64-1.30-1.08-0.87-0.48-0.38-0.710.00-0.44-0.96-0.84-0.36-1.33-1.11 NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.39-1.26-0.38-1.200.00-0.52-1.190.33-1.270.00-0.74-0.38-0.20-0.25-1.31-1.19V-0.20-1.310.64-0.20-0.49-0.21NaN0.30-0.55-0.50-0.44-0.730.00-0.270.00-0.300.000.00-1.21-1.04-0.44-0.38-1.01-0.84-0.51-1.22-1.07-0.27-0.34-0.430.00NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.40-1.07-0.37-0.94-0.38-0.80-0.66-0.24-0.34-0.940.000.00-0.31-1.21-0.22-1.41A-1.09-0.99-0.260.55-0.70-1.23-0.730.550.210.53-0.96-0.21-0.26-0.49-0.480.00-0.48-0.50-1.50-1.220.04-1.23-0.87-0.89-0.77-1.22-0.550.000.00-0.44-0.54NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.02-0.99-0.14-0.44-0.86-0.65-0.90-1.11 -1.05-1.28-0.25-0.32-1.26-0.40-0.82NaNG-0.99-0.20NaN0.00-0.42-1.06-0.120.300.33-0.26-0.45-0.63-0.640.00-0.72-0.79-0.73-0.31-1.41-1.25-0.58-1.21-1.21-0.59-1.12-1.06-0.55-0.16-0.28-0.35-0.51NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.09-1.29-0.540.00-1.10-1.020.00-1.85-0.33-1.09-0.54-0.42-1.59-1.19-0.24NaNC-0.77-0.89-1.030.09-0.32-1.29-0.210.480.65-1.40-1.080.05-0.66-0.21-0.88-0.23-1.02-0.85-1.26-0.35-0.70-1.84-1.19-0.480.14-0.85-0.29-0.10-0.42-0.86-0.75NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.45-1.15-0.53-0.55-0.80-0.94-0.44-0.63-1.47-0.93-0.36-0.71-0.32-1.41-1.03-0.11 S-1.27-0.350.11 0.41-1.12-0.800.000.000.00-0.27-1.07-0.12-1.26-0.34-1.18-0.56-1.16-1.20-0.63-0.890.07-1.26-0.47-0.46-0.42-1.03-0.080.16-0.13-0.95-1.08NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.26-0.93-0.23-0.83-0.99-0.33-0.72-1.05-1.20-1.10-1.01-0.78-0.21-0.41-1.23-0.50T-0.31-0.710.56-0.10-1.13-0.48-0.230.520.590.02-1.430.00-1.15-1.11 -1.15-0.41-1.37-0.92-0.98-1.39-0.01-0.62-0.65-0.74-0.36-0.870.00-0.02-0.24-1.28-1.07NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.61-0.93-0.28-1.17-0.85-0.66-1.17-0.36-1.43-1.22-1.38-1.72-0.81-0.19-0.92-1.02NNaN-1.020.00-0.29-0.37-0.49-0.040.530.560.00-0.38-0.23-1.21-0.94-1.38-1.38-1.13-1.470.00-0.73-0.44-1.00-2.02-0.75-0.82-0.56-0.08-0.30-0.45-0.80-1.26NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.34-0.52-0.34-1.17-1.16-1.25-1.11 -1.01-0.54-1.22-1.16-0.92-1.09-0.96-0.96-0.16Q-1.18-0.560.66-0.28-1.52-1.20NaN-1.620.450.13-1.25-0.66-0.84-0.89-0.82-1.52-1.69-1.65-0.90-1.28-0.41-1.16-0.43-0.83-1.13-0.41-0.770.14-0.31-0.19-1.29NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.33-0.820.00-1.19-0.52-0.59-1.64-0.89-1.38-0.45-0.96-1.01-1.75-0.89-0.38-0.99D-1.37-0.89-0.25-0.330.00-1.12NaN-0.140.600.030.00-1.45-1.09-0.41-1.01-0.62-0.92-0.79-0.47-0.79-0.83-0.94-1.43-0.97-1.28-0.39-0.57-0.30-0.19-0.21-0.59NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.25-1.37-0.56-0.63-0.96-1.28-0.53-1.040.00-0.95-0.25-1.52-1.16-1.03-0.23-0.43E-2.17-1.81-0.95-0.77-0.34-1.20-0.89-0.46-0.12-0.04-0.53-0.88-0.27-1.90-0.48-1.48-0.33-0.46-1.74-1.25-0.25-1.22-1.15-1.05-1.23-1.52-1.03-0.45-0.150.00-1.38NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.10-0.29-0.30-1.04-0.96-1.42-1.23-1.31-0.07-1.12-0.71-0.14-1.14-0.890.00-1.52H-0.91-1.000.00-1.47-0.99-1.15-0.67-1.190.490.04-0.50-0.93-1.54-2.06-1.12-1.07-1.00-0.98-0.85-0.49-0.58-1.50-1.08-0.46-1.060.00-0.68-0.29-0.42-0.72-1.37NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.11 -1.14-0.23-1.13-0.61-1.00-0.99-1.94-0.79-1.43-1.72-1.00-1.19-0.17-1.11 -0.23K-0.42-0.25-0.42-1.03-1.14-1.42-0.480.64-0.33-0.04-1.16-0.83-1.12NaN-1.28-0.97-1.13-1.13-0.52-1.010.00-0.65-1.09-0.70-1.16-0.72-0.620.82-0.27-0.34-1.81NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.580.00-0.07-1.09-1.35-0.95-1.35-0.47-1.03-1.28-0.88-1.14-1.03NaN-0.30NaNR-0.620.00-0.23-0.53-1.30-1.27-0.05-0.49-0.67-0.17-1.40-0.89-1.42-0.54-1.21-1.03-1.20-1.15-1.32-1.45-0.28-1.07-0.820.00-1.22-0.30-0.640.74-0.30-1.17-1.28NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.96-0.45-0.13-0.95-0.91-0.90-0.96-0.48-1.29-0.80-0.87-0.98-1.49-0.40-1.67-0.81AmiE I38V 616263646566676869707172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110 111112 113 114 115 116 117 118 119 120Mutation SLQGIMYDPAEMMETAVAIPGEETEIFSRACRKANVWGVFSLTGERHEEHPRKAPYNTLVSTOP 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-0.27NaN-2.40NaNNaN-1.39-1.49NaN-1.620.000.00-0.45-0.22-0.89-1.85-0.34-1.87NaN-1.720.00NaN-0.16-1.19E-2.14-2.22-0.40-1.05-0.26NaN0.00-1.26-0.39-0.89-0.46NaN-1.52-0.18-3.79-1.46-2.43-1.68-1.57NaNNaN0.00-0.71NaN-2.06NaN-1.96-0.09-0.15-2.49-1.17-2.11 -1.910.00-1.94-1.26-0.22-1.18NaN-0.30-1.26-1.85-2.99-1.33-1.12-1.76-0.05-0.18-1.23-1.57-1.67-2.380.00-2.04-0.62-1.68-0.24-1.32-1.27-1.39H-2.11 -1.42-0.64-0.530.04-1.02-0.03-1.73-1.49-0.86-1.46-0.19-1.61-1.74-1.82-0.28-2.20-1.44-1.52NaN-0.45-1.55-0.09NaN-0.40-1.48-5.09-0.06-0.41-1.83-0.42-2.12-1.79-0.43-2.17-1.67-2.57NaN-1.72-2.20-1.65-2.00-1.46-2.40-2.54-1.68-0.63-0.62-1.89-0.89-0.33-2.04-2.01NaNNaN-1.83-0.62NaN-2.25-1.75K-2.73-2.78-2.88-0.770.38NaN-0.05-1.08-0.34-0.940.00NaN-0.400.00-5.31-2.24-2.68-1.34-3.29NaN-0.76-0.46-0.13-1.02-2.82NaN-3.380.26-0.09-1.10-1.91-2.05-1.82-0.20-1.76NaN0.00NaN-0.080.00NaN-6.23-1.30-1.52-1.21-1.98-2.45-2.17-2.96-0.16-2.09-1.08-0.36-2.85-2.01-0.65NaNNaN-2.33-0.40R-1.64-1.78-1.32-0.800.10-0.630.02-2.730.04-0.69-0.49NaN0.00-0.47-1.94-1.84-1.17-0.70-0.60-0.51-0.96-1.530.05-0.20-1.63-0.77-1.13-0.11 0.06-1.44-1.73-1.76-1.82-0.57-1.44-0.82-0.32-0.80-0.36-0.31-2.42-2.29-3.59-1.59-1.02-0.55-2.15NaN-1.11 -1.55-1.75-1.39-1.76-1.65-0.350.00-2.12-0.29-1.83-0.62AmiE I38V 181182183184185186187188189190191192193194195196197198199200201202203204205206207208209210211 212213214215216217218219220221222223224225226227228229230231232233234235236237238239240Mutation KGAELIVRCQGYMYPAKDQQVMMAKAMAWANNCYVAVANAAGFDGVYSYFGHSAIIGFDGSTOP *-0.65-1.99-2.11 -0.31-1.10-1.38-1.96-2.17-0.50-0.08NaN-0.55-2.48-0.22NaN-1.60-0.36-2.14-0.28-0.74-2.19-1.37-3.36NaN-0.53-1.17NaN-1.28-0.16-1.76-1.34-3.63-0.48-0.45-1.40-1.65-1.26-1.76NaNNaN-1.68NaN-1.74-3.18-1.78-1.93-0.12-2.28-0.25-2.00NaN-2.44-1.82-1.74-2.09-2.11 -5.18-1.68-1.03-3.48F-1.91-7.63-1.71-1.65-0.40-0.16-0.44-1.44-0.34-1.09NaN-0.25NaN-0.39NaN-1.20-1.14-1.22NaN-1.51-0.18-1.20-1.53-2.12NaNNaN-0.99-1.40-0.64-1.24-3.98-1.76-0.41-0.71-0.43-0.98-1.26NaNNaN-0.67-1.68-2.740.00-1.32-1.58-0.09-0.08-1.190.320.00NaN-2.25-2.85-1.38-0.74-0.37NaN0.00-1.44-1.30W-2.37-1.88-2.46NaN-0.73NaNNaN-0.84-0.62NaNNaN-2.38-1.48-1.04NaN-1.11 NaN-1.67NaN-1.76-0.56-0.74NaN-1.09NaNNaNNaNNaN0.00-1.21-1.95NaN-0.93-1.48-1.21-1.32NaNNaNNaNNaNNaN-1.23-0.84NaNNaNNaNNaNNaNNaN-0.30NaN-1.72-1.04-0.99-1.65-1.60-1.87NaNNaNNaNY-2.41-1.76-3.70NaN-2.41-1.90-1.87-1.46-0.310.13NaN0.00-2.930.00-0.90-1.53NaN-0.46-1.85-1.75-0.93-1.17-1.55-1.47-2.36NaN-1.34-0.87-0.85-2.20-0.40-0.79-0.280.00-3.54NaN-1.49NaN-0.35NaN-2.25-1.55-0.25-0.28NaN-0.590.00-0.760.000.23NaN-0.18-1.48-1.51-1.730.64-1.58-0.68-0.32-2.15P-2.12-1.74-1.51-2.14-1.83-1.95-1.52-0.75-1.30-0.07-3.02-1.830.53-1.540.00-1.03-1.53-1.18-0.87-0.87-1.39-1.65-1.59-0.92-2.01-0.93-1.66-0.46-1.63-1.41-1.77-1.93-1.44-1.76-2.61-0.86-2.09-0.73-2.66-0.61-0.94-1.80-2.00-2.16-2.23-2.02NaN-1.51-1.62-1.60NaN-0.56-2.02-1.54-1.75-2.12-1.93-2.25-1.73-1.95START M-0.53-2.27-1.13NaN-1.25-0.40-1.50NaN-1.27NaNNaN-1.280.00-1.32NaN-1.32-0.36-1.13-0.43-2.671.040.000.00NaN-0.20-0.820.00NaNNaN-1.00NaN0.46NaN-1.09NaNNaN-0.17NaN-1.04NaNNaN-2.08-1.43-1.52NaN-0.91NaNNaNNaN-0.56NaNNaNNaN0.88-0.56-0.64NaNNaN-1.19NaNI-1.78-3.05-1.88NaN-1.630.00-0.30NaN-2.29-0.91-1.64-1.96-0.25-2.00-2.15-1.50-0.29-1.35-0.25-2.030.71-0.27-0.21-1.80-1.25-1.19-0.20-1.10NaN-2.31-0.35-0.28NaN-1.49-0.36-1.72-0.44-1.98-0.54-0.95-1.63-1.77-1.42-1.87NaN-0.22-1.22-0.36-1.06-0.61NaN-1.40-0.88-0.670.000.00-1.47-1.02-1.98-1.87L-1.58-2.20-2.61-1.960.00-1.14-1.05-0.52-2.89-0.18-3.73-1.90-0.50-1.55-0.33-2.32-1.13-1.31-0.57-1.150.85-0.08-0.49-1.85-1.52-2.07-0.59NaN-0.35-2.35-2.160.03-3.51-4.37-1.11 -2.58-0.59-2.35-2.27-1.04-1.66-2.60-0.65-1.44NaN-0.57-2.27-2.16-1.65-0.27-1.34-0.58-2.54-0.64-1.03-0.98-1.77-0.53-1.91-1.71V-1.86-0.76-0.41-0.26-1.10-0.310.00NaN-1.70-1.48-0.30-2.11 -0.40-3.10-1.32-0.20-0.57-0.47-0.67-1.820.00-0.54-0.28-0.33-2.12-0.06-0.48-0.35-2.20-0.34-1.740.190.52NaN0.00-0.360.00-0.18NaN-0.30-0.36-1.08-0.73-0.20-0.140.00NaN-0.65-1.55-0.93-0.18-1.57-1.94-0.50-0.71-0.65-0.89-1.05-0.14-0.40A-1.83-1.230.00-1.03-2.61-1.15-0.53-2.02-0.390.39-0.52-1.44-0.43-0.15-0.950.00-0.42-0.90-0.40-1.21-0.37-0.11 -2.040.00-2.800.00-1.700.00-2.990.00-1.92-1.08-2.16-2.20-0.550.00-0.540.00-1.300.000.00-1.57-1.48-0.93-0.74-0.37-1.330.15-1.88-1.84-0.38-1.32-3.090.00-1.79-1.96-0.91-2.12-0.50-0.77G-1.660.00-0.94-0.30-1.99-1.46-0.86-0.56-0.79-0.320.00-3.46-0.50-2.23-1.81-0.32-2.23-0.51-2.67-1.56-0.85-1.12-1.26-0.44-1.40-0.46-2.57-0.35-0.70-0.73-1.74-0.75-0.41-2.11 -0.89-0.80-1.23-0.65NaN-0.03-0.750.00-2.24-0.270.00-0.12NaN-0.20NaN-1.180.00-0.61-0.64-0.26-1.28-4.060.00-1.48-0.220.00C-2.27-0.35-1.77-1.24-1.48-1.73-0.50-0.320.00NaN-0.39-0.48-0.45-0.33NaN0.020.30-1.22-0.75-1.96-0.51-0.50-1.68-0.08-1.900.10-1.93NaN-0.36-0.44-2.210.510.00-0.41-1.68-0.90-0.53NaN-1.68NaNNaN-0.61-0.67-1.10-0.13-0.55-0.11 -0.26-0.100.25-0.12-0.83-0.530.77-2.31-1.32-0.41-1.06-0.74-0.21S-1.84-1.19-0.72-5.04-0.75-0.65-1.57-0.61-0.310.84-0.46-1.22-0.47-0.08-0.44-0.22-1.07-1.15-0.99-1.04-0.56-0.91-2.770.00-2.00-0.35-2.76-0.35-0.93-0.93-0.64-0.40-0.57-1.88-1.87-0.56-1.90-0.43-0.40-0.21-0.14-1.31-0.53-3.20-0.28-1.09-0.690.00-0.66-0.21-0.23-1.300.00-0.50-1.25-0.970.10-0.83-2.75-0.40T-1.27-1.80-0.65-1.93-1.94-0.55-1.41-1.48-1.770.37NaN-1.74-0.39-1.15-0.14-0.52-1.04-1.09-0.19-1.46-0.19-0.23-0.47-0.47-0.98-0.41-0.52-0.29-2.41-0.78-1.140.40-0.80-1.46-2.06-0.50-0.92-0.51-0.58-0.300.41-1.43-0.97-2.42-1.23-0.29-1.44-0.38-1.48-0.70NaN-1.28-1.20-0.36-1.02-0.86-1.48-1.97NaN-2.45N-0.61-1.44NaN-2.31-2.35-0.27-1.68-1.52-1.58-0.54-1.90-1.06-0.95-0.29-0.84-0.42-0.35-0.41-1.35-0.750.250.00-1.090.26-0.35-2.07-1.46-1.93NaNNaN0.000.00-2.09-1.13-3.18NaN-2.31-1.680.00-1.36-1.98-1.31-0.41-0.39-1.26-1.81-0.46-0.31-0.17-1.31NaN-0.24-0.63-1.80-0.77-0.42-0.77-1.97-0.18NaNQ-0.71-2.03-1.83-0.42-2.14-1.49-1.29NaN-2.540.00-1.40-1.55-0.78-0.45NaN-0.95-0.50-0.800.000.00-0.17-0.83NaN-1.52-0.85-1.85-1.02-1.14-2.15-1.41-1.430.76-2.25-2.99-3.10NaN-0.30-1.93NaNNaN-2.83-1.86-1.85-1.72NaN-0.78-1.36-1.13-0.98NaNNaN-0.32-1.43-0.28-2.95-1.78-2.02NaN-1.84-1.73D-2.02-0.68-1.60-0.33-6.50-2.16-0.53-1.07-2.12-0.47-0.51-1.02NaN-0.88NaN-0.43-1.360.00-2.28-1.91-0.78-1.47-1.72-0.29-1.40-1.79NaN-0.37-1.68-1.79-0.41-0.34-1.81-1.58-0.45-0.44-2.31-2.44-0.30-0.19NaN-0.87-2.040.00-0.41-0.59-0.24-1.36-0.09NaN-0.29-0.67-2.61-0.71-2.23-1.54-0.42-1.360.00-0.52E-0.67-1.53-0.520.00-1.80NaN-1.95NaN-3.02-0.09-0.80-2.00NaN-1.26NaN-1.41-0.11 0.25-0.63-0.86-1.58-1.51NaN-2.12-0.17-0.30NaNNaNNaN-0.47-1.28-0.75NaN-1.32-1.81-1.58-0.72-0.28NaN-1.32-0.26-4.44-1.63-0.24-0.93-1.44-1.52-1.64NaN-1.95NaN-1.28-2.35-1.61-2.49NaN-1.35-1.08-0.17-1.98H-1.92-2.36-3.02-1.42-2.46-1.79-1.63-0.27-1.61-0.26-1.05-0.51-1.33-0.41-0.19-1.98NaN-0.52-0.32-0.79-0.35-1.32-1.36-1.88-1.38-3.07-2.80-1.40-1.30-1.62-1.42-0.16-2.57-0.62-1.76NaN-2.07-2.62-0.79NaN-1.81-1.48-0.24-0.79-1.52-0.67-0.08-0.05-0.26-0.30NaN0.00-1.94-1.06-2.15-2.22-1.89-0.99-0.50-1.65K0.00-2.97-2.23-0.40-1.44NaN-1.63-1.84NaN-0.04NaN-1.71-0.53-2.25NaN-2.590.00-1.75-0.30-0.80-0.67-0.83-0.25-1.620.00NaN-0.42-1.58-1.00-1.49-0.390.07-1.36-2.41NaN-1.40-2.14-2.41-0.30NaN-2.27-1.19-2.17-1.17-1.01-0.81-1.44-0.30NaNNaN-1.48-2.83-3.03-1.52-2.17-3.37NaN-2.83-0.87-1.79R-0.31-1.47-1.61-4.18-1.87-1.97-1.920.00-0.49-0.14-0.97-2.56-1.20-1.62-0.83-1.65-0.20-1.30-0.33-0.80-0.11 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NaN-0.28NaNNaN0.17-0.14-0.27-0.65-0.01-0.16NaNNaN-0.29-0.59-0.48-0.51-0.65-0.34-0.39-0.62-0.71-0.32-0.31-0.10-0.14-0.660.37-0.46-0.18I-1.86-0.56-1.22-0.54-1.51-0.71-0.27NaN-0.05-0.27NaN-1.62-0.86-0.98-2.93-0.34-0.51-0.56-0.08-0.53-0.020.14-0.18NaN-0.51-0.24-0.24-0.39-0.40-0.33-0.41-0.31-0.96-0.07-0.38-0.08-0.10-0.550.16-0.14-0.28L-0.32-0.51-1.38-0.51-1.47-0.69-0.24-0.17-0.990.08-1.53-1.75-0.91-0.47-1.83-1.35-0.35-0.65-0.62-0.76-0.310.00-1.59-0.45-0.59-0.86-0.14-0.48-0.53-0.34-0.33-0.42-0.39-0.20-0.31-0.160.00-0.270.12-0.06-0.18V-1.52-1.21-0.53-0.58-1.96-0.99-0.57-0.790.00-0.72-0.24-0.41-0.10-0.86-0.23-0.89-0.15-0.810.00-0.36-0.65-0.48-1.63NaN-0.21-0.560.01-0.260.00-0.25-0.24-0.69-0.810.00-0.17-0.05-0.14-0.690.12-0.24-0.21A-0.67-1.68-1.13-1.67-1.36-1.64-0.44-1.28-0.43-0.29-0.790.00-0.38-0.120.00-1.31-0.20-0.15-0.44-0.39-0.05-1.46-0.22-1.140.00-0.26-0.07-0.06-0.060.00-0.04-0.45-0.23-0.07-0.020.050.080.020.19-0.04-0.08G-1.66-1.65-0.36-2.53-1.29-0.87-1.36-0.11 -0.41-0.93-0.26-0.69-0.19-0.79-0.50-0.30-0.31-1.03-0.45-0.31-0.48-2.29-1.28-0.570.02-0.41-0.860.00-0.16-0.08-0.13-0.31-0.44-0.160.000.220.05-0.190.300.050.00C-0.97-0.56-1.19-1.23-0.19-0.27-0.65-0.09-1.79-0.45-1.07-0.30NaN-0.83-0.08-1.89-0.69-0.53-0.54-0.59-0.16-2.050.01-0.30-0.22-0.47-0.37-0.30-0.24-0.28-0.260.00-0.49-0.070.50-0.04-0.13-0.680.13-0.24-0.14S-0.37-0.63-0.91-0.65-0.71-0.68-0.71-0.65-1.35-0.13-2.20-0.36-0.87-0.64-0.58-0.46-0.34-0.43-0.90-1.86-0.43-1.61-0.11 -0.570.00-0.07-0.16-0.200.100.15-0.02-0.37-0.17-0.16-0.160.17-0.06-0.200.20-0.20-0.11 T-0.45-2.02-1.06-1.44-2.27-0.970.00-2.12-1.320.00-1.53-0.61-0.63-0.39-0.57-0.84-0.27-0.20-1.24-0.91-0.34-1.800.00-0.98-0.080.000.00-0.160.060.01-0.13-0.42-0.25-0.03-0.220.13-0.11 -0.200.22-0.22-0.24NNaN-1.44-2.03-1.66-0.35-0.30-0.14-0.85-1.230.04-0.06-2.20-0.27-0.48-2.53-1.76-0.610.00-2.47-0.91-0.28-1.70-0.18NaN-0.01-0.05-1.24-0.21-0.26-0.05-0.09-0.210.25-0.25-0.100.21-0.05-0.340.28-0.07-0.14Q-0.11 -1.72-0.42-2.03-1.55-0.84-0.39-1.23NaN-0.01-0.65-1.65-0.26-0.34-1.31-1.03-0.140.22-2.05-0.32-0.12-0.59-1.45-1.950.06-0.21-0.86-0.11 -0.550.010.00-0.34-0.10-0.22-0.090.250.04-0.220.20-0.15-0.23DNaN-0.92-0.07-1.75-0.70-1.00-0.99-1.23-0.24-1.010.00-0.53-0.11 NaN-2.20-1.41-0.27-0.23NaN-0.53-1.00-1.53NaN-0.95-0.37-0.34-1.17-0.11 -0.23-0.010.05-0.18-0.40-0.010.050.380.25-0.380.580.020.14ENaN-1.330.00-1.36NaN-2.00-0.73NaN-1.18-0.48-0.08-2.020.00-0.22-0.41-1.100.00-0.65-0.330.00-0.96-1.47NaNNaN-0.39-0.27-0.83-0.10-0.560.120.12-0.12-0.12-0.11 0.040.290.18-0.260.350.000.07H-1.02-1.81-1.19-1.91-0.26-0.30-1.07NaN-1.92-0.14-0.51-0.25-0.71-0.89-1.55-1.16-0.64-0.11 -1.50-1.24-0.22-1.57NaN-0.32-0.19-0.21-1.26-0.25-0.67-0.20-0.17-0.36-0.44-0.12-0.260.04-0.08-0.450.16-0.35-0.23KNaN-1.19-0.30NaN-1.76-0.03-1.23-0.77NaN-0.28-0.54-1.30-0.220.00-1.95-0.55-0.13-0.44NaN-0.370.03-1.74NaNNaN0.07-0.20-0.53-0.22-0.640.02-0.07-0.40-0.15-0.35-0.160.19-0.15-0.280.070.150.04R-0.71-1.62-1.80-1.23-1.630.00-1.42-0.19NaN-0.27-1.70-1.86-1.07-0.12-2.520.00-0.38-0.55-1.29-0.830.00-0.72-1.660.00-0.060.06-0.96-0.33-0.800.10-0.32-0.400.01-0.39-0.230.00-0.11 -0.35-0.02-0.05-0.10Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged AmiE I38V 123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960Mutation MRHGDISSSNDTVGVAVVNYKMPRLHTAAEVLDNARKVAEMIVGMKQGLPGMDLVVFPEYSTOP *-1.16-0.16-1.42-1.15-1.09-1.28NaN-0.27-0.09-1.18-1.38-1.12-1.38-0.88-1.40-1.16-1.34-2.15-1.37-0.38-0.58-1.04-1.22-1.06-0.88-1.21-1.31-1.09-1.20-0.41-1.06NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.24-0.42-0.62-1.08-1.40-1.06-1.02-1.36-0.88-0.98-1.22-1.24-1.07-1.28-0.24-0.17F-0.76-1.45-0.96-1.48-1.28-0.35NaN-0.840.43-0.92-1.17-1.37-1.96-1.09-0.92-1.82-1.13-1.46-2.05-0.26-1.38-1.64-1.25-1.31-0.41-1.03-1.31-1.56-0.67-1.17-0.39NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.05-1.07-0.72-1.07-0.01-1.23-0.92-0.95-0.70-0.96-0.30NaN0.00NaNNaN-0.21WNaN-1.09NaN-0.96-1.37-0.77NaN-0.28NaNNaNNaNNaNNaN-0.96-1.17-1.62-1.28-1.29-0.83-0.45-1.24-2.16-1.17-2.20-1.24-0.96-1.16-1.28-0.87-1.31-1.22NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.05-1.31-0.89-1.21-0.09-1.22NaN-1.19NaN-0.77-1.24-0.93NaNNaN-0.82NaNY-1.41-1.28-0.12-0.14-0.36-0.95NaN-0.780.03-0.39-0.20-1.62-1.27-1.41-1.73-1.08-1.28-1.18-0.410.00-1.05-1.24-0.83-1.14-0.99-0.19-0.88-1.30-0.60-1.67-1.22NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.32-1.08-0.57-0.910.34-1.58-1.11 -1.16-0.32-1.28-1.31-1.24-0.22-0.82-1.170.00P-1.12-1.04-0.56-1.23-1.14-1.54-0.79-0.38-0.38-0.02-1.30-0.82-1.17-0.83-1.60-0.98-1.24-1.09-1.26-1.190.38-1.160.00-0.89-1.24-0.69-0.810.26-0.33-0.98-1.19NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.31-1.26-0.84-1.18-0.280.00-1.29-1.17-1.11 -0.58-1.48-1.87-1.640.00-0.95-0.97START M0.00-0.82-0.32NaN-1.07-0.50NaN-0.850.58-0.31NaNNaN-1.52NaN-1.75-0.82-0.34-0.50-0.91-0.99-0.370.00-0.97-0.92-0.79-1.21-1.84-0.54-0.41-0.77-1.12NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN0.00-0.33-0.45-1.140.03-1.82-1.120.00-1.80-0.27NaN-0.12NaNNaN-0.89NaNI-0.11 -0.230.61-0.33-1.370.00-0.050.350.49-0.29-1.14-0.35-0.23-0.78-0.41-0.98-1.19-0.78-0.54-1.00-0.88-0.09-0.85-0.94-0.12-0.82-0.52-0.64-0.35-1.28-0.32NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.23-1.09-0.42-1.31-0.05-1.06-1.25-0.22-1.32-0.95-0.27-0.08-0.19-1.33-1.38-0.80L-0.25-2.500.05-1.14-1.13-0.44NaN-0.360.24-0.29-1.04-1.17-0.33-1.58-0.49-1.18-0.94-0.64-1.30-1.08-0.87-0.48-0.38-0.710.00-0.44-0.96-0.84-0.36-1.33-1.11 NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.39-1.26-0.38-1.200.00-0.52-1.190.33-1.270.00-0.74-0.38-0.20-0.25-1.31-1.19V-0.20-1.310.64-0.20-0.49-0.21NaN0.30-0.55-0.50-0.44-0.730.00-0.270.00-0.300.000.00-1.21-1.04-0.44-0.38-1.01-0.84-0.51-1.22-1.07-0.27-0.34-0.430.00NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.40-1.07-0.37-0.94-0.38-0.80-0.66-0.24-0.34-0.940.000.00-0.31-1.21-0.22-1.41A-1.09-0.99-0.260.55-0.70-1.23-0.730.550.210.53-0.96-0.21-0.26-0.49-0.480.00-0.48-0.50-1.50-1.220.04-1.23-0.87-0.89-0.77-1.22-0.550.000.00-0.44-0.54NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.02-0.99-0.14-0.44-0.86-0.65-0.90-1.11 -1.05-1.28-0.25-0.32-1.26-0.40-0.82NaNG-0.99-0.20NaN0.00-0.42-1.06-0.120.300.33-0.26-0.45-0.63-0.640.00-0.72-0.79-0.73-0.31-1.41-1.25-0.58-1.21-1.21-0.59-1.12-1.06-0.55-0.16-0.28-0.35-0.51NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.09-1.29-0.540.00-1.10-1.020.00-1.85-0.33-1.09-0.54-0.42-1.59-1.19-0.24NaNC-0.77-0.89-1.030.09-0.32-1.29-0.210.480.65-1.40-1.080.05-0.66-0.21-0.88-0.23-1.02-0.85-1.26-0.35-0.70-1.84-1.19-0.480.14-0.85-0.29-0.10-0.42-0.86-0.75NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.45-1.15-0.53-0.55-0.80-0.94-0.44-0.63-1.47-0.93-0.36-0.71-0.32-1.41-1.03-0.11 S-1.27-0.350.11 0.41-1.12-0.800.000.000.00-0.27-1.07-0.12-1.26-0.34-1.18-0.56-1.16-1.20-0.63-0.890.07-1.26-0.47-0.46-0.42-1.03-0.080.16-0.13-0.95-1.08NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.26-0.93-0.23-0.83-0.99-0.33-0.72-1.05-1.20-1.10-1.01-0.78-0.21-0.41-1.23-0.50T-0.31-0.710.56-0.10-1.13-0.48-0.230.520.590.02-1.430.00-1.15-1.11 -1.15-0.41-1.37-0.92-0.98-1.39-0.01-0.62-0.65-0.74-0.36-0.870.00-0.02-0.24-1.28-1.07NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.61-0.93-0.28-1.17-0.85-0.66-1.17-0.36-1.43-1.22-1.38-1.72-0.81-0.19-0.92-1.02NNaN-1.020.00-0.29-0.37-0.49-0.040.530.560.00-0.38-0.23-1.21-0.94-1.38-1.38-1.13-1.470.00-0.73-0.44-1.00-2.02-0.75-0.82-0.56-0.08-0.30-0.45-0.80-1.26NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.34-0.52-0.34-1.17-1.16-1.25-1.11 -1.01-0.54-1.22-1.16-0.92-1.09-0.96-0.96-0.16Q-1.18-0.560.66-0.28-1.52-1.20NaN-1.620.450.13-1.25-0.66-0.84-0.89-0.82-1.52-1.69-1.65-0.90-1.28-0.41-1.16-0.43-0.83-1.13-0.41-0.770.14-0.31-0.19-1.29NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.33-0.820.00-1.19-0.52-0.59-1.64-0.89-1.38-0.45-0.96-1.01-1.75-0.89-0.38-0.99D-1.37-0.89-0.25-0.330.00-1.12NaN-0.140.600.030.00-1.45-1.09-0.41-1.01-0.62-0.92-0.79-0.47-0.79-0.83-0.94-1.43-0.97-1.28-0.39-0.57-0.30-0.19-0.21-0.59NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.25-1.37-0.56-0.63-0.96-1.28-0.53-1.040.00-0.95-0.25-1.52-1.16-1.03-0.23-0.43E-2.17-1.81-0.95-0.77-0.34-1.20-0.89-0.46-0.12-0.04-0.53-0.88-0.27-1.90-0.48-1.48-0.33-0.46-1.74-1.25-0.25-1.22-1.15-1.05-1.23-1.52-1.03-0.45-0.150.00-1.38NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.10-0.29-0.30-1.04-0.96-1.42-1.23-1.31-0.07-1.12-0.71-0.14-1.14-0.890.00-1.52H-0.91-1.000.00-1.47-0.99-1.15-0.67-1.190.490.04-0.50-0.93-1.54-2.06-1.12-1.07-1.00-0.98-0.85-0.49-0.58-1.50-1.08-0.46-1.060.00-0.68-0.29-0.42-0.72-1.37NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-1.11 -1.14-0.23-1.13-0.61-1.00-0.99-1.94-0.79-1.43-1.72-1.00-1.19-0.17-1.11 -0.23K-0.42-0.25-0.42-1.03-1.14-1.42-0.480.64-0.33-0.04-1.16-0.83-1.12NaN-1.28-0.97-1.13-1.13-0.52-1.010.00-0.65-1.09-0.70-1.16-0.72-0.620.82-0.27-0.34-1.81NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.580.00-0.07-1.09-1.35-0.95-1.35-0.47-1.03-1.28-0.88-1.14-1.03NaN-0.30NaNR-0.620.00-0.23-0.53-1.30-1.27-0.05-0.49-0.67-0.17-1.40-0.89-1.42-0.54-1.21-1.03-1.20-1.15-1.32-1.45-0.28-1.07-0.820.00-1.22-0.30-0.640.74-0.30-1.17-1.28NaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaNNaN-0.96-0.45-0.13-0.95-0.91-0.90-0.96-0.48-1.29-0.80-0.87-0.98-1.49-0.40-1.67-0.81AmiE I38V 616263646566676869707172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110 111112 113 114 115 116 117 118 119 120Mutation SLQGIMYDPAEMMETAVAIPGEETEIFSRACRKANVWGVFSLTGERHEEHPRKAPYNTLVSTOP 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-0.27NaN-2.40NaNNaN-1.39-1.49NaN-1.620.000.00-0.45-0.22-0.89-1.85-0.34-1.87NaN-1.720.00NaN-0.16-1.19E-2.14-2.22-0.40-1.05-0.26NaN0.00-1.26-0.39-0.89-0.46NaN-1.52-0.18-3.79-1.46-2.43-1.68-1.57NaNNaN0.00-0.71NaN-2.06NaN-1.96-0.09-0.15-2.49-1.17-2.11 -1.910.00-1.94-1.26-0.22-1.18NaN-0.30-1.26-1.85-2.99-1.33-1.12-1.76-0.05-0.18-1.23-1.57-1.67-2.380.00-2.04-0.62-1.68-0.24-1.32-1.27-1.39H-2.11 -1.42-0.64-0.530.04-1.02-0.03-1.73-1.49-0.86-1.46-0.19-1.61-1.74-1.82-0.28-2.20-1.44-1.52NaN-0.45-1.55-0.09NaN-0.40-1.48-5.09-0.06-0.41-1.83-0.42-2.12-1.79-0.43-2.17-1.67-2.57NaN-1.72-2.20-1.65-2.00-1.46-2.40-2.54-1.68-0.63-0.62-1.89-0.89-0.33-2.04-2.01NaNNaN-1.83-0.62NaN-2.25-1.75K-2.73-2.78-2.88-0.770.38NaN-0.05-1.08-0.34-0.940.00NaN-0.400.00-5.31-2.24-2.68-1.34-3.29NaN-0.76-0.46-0.13-1.02-2.82NaN-3.380.26-0.09-1.10-1.91-2.05-1.82-0.20-1.76NaN0.00NaN-0.080.00NaN-6.23-1.30-1.52-1.21-1.98-2.45-2.17-2.96-0.16-2.09-1.08-0.36-2.85-2.01-0.65NaNNaN-2.33-0.40R-1.64-1.78-1.32-0.800.10-0.630.02-2.730.04-0.69-0.49NaN0.00-0.47-1.94-1.84-1.17-0.70-0.60-0.51-0.96-1.530.05-0.20-1.63-0.77-1.13-0.11 0.06-1.44-1.73-1.76-1.82-0.57-1.44-0.82-0.32-0.80-0.36-0.31-2.42-2.29-3.59-1.59-1.02-0.55-2.15NaN-1.11 -1.55-1.75-1.39-1.76-1.65-0.350.00-2.12-0.29-1.83-0.62AmiE I38V 181182183184185186187188189190191192193194195196197198199200201202203204205206207208209210211 212213214215216217218219220221222223224225226227228229230231232233234235236237238239240Mutation KGAELIVRCQGYMYPAKDQQVMMAKAMAWANNCYVAVANAAGFDGVYSYFGHSAIIGFDGSTOP *-0.65-1.99-2.11 -0.31-1.10-1.38-1.96-2.17-0.50-0.08NaN-0.55-2.48-0.22NaN-1.60-0.36-2.14-0.28-0.74-2.19-1.37-3.36NaN-0.53-1.17NaN-1.28-0.16-1.76-1.34-3.63-0.48-0.45-1.40-1.65-1.26-1.76NaNNaN-1.68NaN-1.74-3.18-1.78-1.93-0.12-2.28-0.25-2.00NaN-2.44-1.82-1.74-2.09-2.11 -5.18-1.68-1.03-3.48F-1.91-7.63-1.71-1.65-0.40-0.16-0.44-1.44-0.34-1.09NaN-0.25NaN-0.39NaN-1.20-1.14-1.22NaN-1.51-0.18-1.20-1.53-2.12NaNNaN-0.99-1.40-0.64-1.24-3.98-1.76-0.41-0.71-0.43-0.98-1.26NaNNaN-0.67-1.68-2.740.00-1.32-1.58-0.09-0.08-1.190.320.00NaN-2.25-2.85-1.38-0.74-0.37NaN0.00-1.44-1.30W-2.37-1.88-2.46NaN-0.73NaNNaN-0.84-0.62NaNNaN-2.38-1.48-1.04NaN-1.11 NaN-1.67NaN-1.76-0.56-0.74NaN-1.09NaNNaNNaNNaN0.00-1.21-1.95NaN-0.93-1.48-1.21-1.32NaNNaNNaNNaNNaN-1.23-0.84NaNNaNNaNNaNNaNNaN-0.30NaN-1.72-1.04-0.99-1.65-1.60-1.87NaNNaNNaNY-2.41-1.76-3.70NaN-2.41-1.90-1.87-1.46-0.310.13NaN0.00-2.930.00-0.90-1.53NaN-0.46-1.85-1.75-0.93-1.17-1.55-1.47-2.36NaN-1.34-0.87-0.85-2.20-0.40-0.79-0.280.00-3.54NaN-1.49NaN-0.35NaN-2.25-1.55-0.25-0.28NaN-0.590.00-0.760.000.23NaN-0.18-1.48-1.51-1.730.64-1.58-0.68-0.32-2.15P-2.12-1.74-1.51-2.14-1.83-1.95-1.52-0.75-1.30-0.07-3.02-1.830.53-1.540.00-1.03-1.53-1.18-0.87-0.87-1.39-1.65-1.59-0.92-2.01-0.93-1.66-0.46-1.63-1.41-1.77-1.93-1.44-1.76-2.61-0.86-2.09-0.73-2.66-0.61-0.94-1.80-2.00-2.16-2.23-2.02NaN-1.51-1.62-1.60NaN-0.56-2.02-1.54-1.75-2.12-1.93-2.25-1.73-1.95START M-0.53-2.27-1.13NaN-1.25-0.40-1.50NaN-1.27NaNNaN-1.280.00-1.32NaN-1.32-0.36-1.13-0.43-2.671.040.000.00NaN-0.20-0.820.00NaNNaN-1.00NaN0.46NaN-1.09NaNNaN-0.17NaN-1.04NaNNaN-2.08-1.43-1.52NaN-0.91NaNNaNNaN-0.56NaNNaNNaN0.88-0.56-0.64NaNNaN-1.19NaNI-1.78-3.05-1.88NaN-1.630.00-0.30NaN-2.29-0.91-1.64-1.96-0.25-2.00-2.15-1.50-0.29-1.35-0.25-2.030.71-0.27-0.21-1.80-1.25-1.19-0.20-1.10NaN-2.31-0.35-0.28NaN-1.49-0.36-1.72-0.44-1.98-0.54-0.95-1.63-1.77-1.42-1.87NaN-0.22-1.22-0.36-1.06-0.61NaN-1.40-0.88-0.670.000.00-1.47-1.02-1.98-1.87L-1.58-2.20-2.61-1.960.00-1.14-1.05-0.52-2.89-0.18-3.73-1.90-0.50-1.55-0.33-2.32-1.13-1.31-0.57-1.150.85-0.08-0.49-1.85-1.52-2.07-0.59NaN-0.35-2.35-2.160.03-3.51-4.37-1.11 -2.58-0.59-2.35-2.27-1.04-1.66-2.60-0.65-1.44NaN-0.57-2.27-2.16-1.65-0.27-1.34-0.58-2.54-0.64-1.03-0.98-1.77-0.53-1.91-1.71V-1.86-0.76-0.41-0.26-1.10-0.310.00NaN-1.70-1.48-0.30-2.11 -0.40-3.10-1.32-0.20-0.57-0.47-0.67-1.820.00-0.54-0.28-0.33-2.12-0.06-0.48-0.35-2.20-0.34-1.740.190.52NaN0.00-0.360.00-0.18NaN-0.30-0.36-1.08-0.73-0.20-0.140.00NaN-0.65-1.55-0.93-0.18-1.57-1.94-0.50-0.71-0.65-0.89-1.05-0.14-0.40A-1.83-1.230.00-1.03-2.61-1.15-0.53-2.02-0.390.39-0.52-1.44-0.43-0.15-0.950.00-0.42-0.90-0.40-1.21-0.37-0.11 -2.040.00-2.800.00-1.700.00-2.990.00-1.92-1.08-2.16-2.20-0.550.00-0.540.00-1.300.000.00-1.57-1.48-0.93-0.74-0.37-1.330.15-1.88-1.84-0.38-1.32-3.090.00-1.79-1.96-0.91-2.12-0.50-0.77G-1.660.00-0.94-0.30-1.99-1.46-0.86-0.56-0.79-0.320.00-3.46-0.50-2.23-1.81-0.32-2.23-0.51-2.67-1.56-0.85-1.12-1.26-0.44-1.40-0.46-2.57-0.35-0.70-0.73-1.74-0.75-0.41-2.11 -0.89-0.80-1.23-0.65NaN-0.03-0.750.00-2.24-0.270.00-0.12NaN-0.20NaN-1.180.00-0.61-0.64-0.26-1.28-4.060.00-1.48-0.220.00C-2.27-0.35-1.77-1.24-1.48-1.73-0.50-0.320.00NaN-0.39-0.48-0.45-0.33NaN0.020.30-1.22-0.75-1.96-0.51-0.50-1.68-0.08-1.900.10-1.93NaN-0.36-0.44-2.210.510.00-0.41-1.68-0.90-0.53NaN-1.68NaNNaN-0.61-0.67-1.10-0.13-0.55-0.11 -0.26-0.100.25-0.12-0.83-0.530.77-2.31-1.32-0.41-1.06-0.74-0.21S-1.84-1.19-0.72-5.04-0.75-0.65-1.57-0.61-0.310.84-0.46-1.22-0.47-0.08-0.44-0.22-1.07-1.15-0.99-1.04-0.56-0.91-2.770.00-2.00-0.35-2.76-0.35-0.93-0.93-0.64-0.40-0.57-1.88-1.87-0.56-1.90-0.43-0.40-0.21-0.14-1.31-0.53-3.20-0.28-1.09-0.690.00-0.66-0.21-0.23-1.300.00-0.50-1.25-0.970.10-0.83-2.75-0.40T-1.27-1.80-0.65-1.93-1.94-0.55-1.41-1.48-1.770.37NaN-1.74-0.39-1.15-0.14-0.52-1.04-1.09-0.19-1.46-0.19-0.23-0.47-0.47-0.98-0.41-0.52-0.29-2.41-0.78-1.140.40-0.80-1.46-2.06-0.50-0.92-0.51-0.58-0.300.41-1.43-0.97-2.42-1.23-0.29-1.44-0.38-1.48-0.70NaN-1.28-1.20-0.36-1.02-0.86-1.48-1.97NaN-2.45N-0.61-1.44NaN-2.31-2.35-0.27-1.68-1.52-1.58-0.54-1.90-1.06-0.95-0.29-0.84-0.42-0.35-0.41-1.35-0.750.250.00-1.090.26-0.35-2.07-1.46-1.93NaNNaN0.000.00-2.09-1.13-3.18NaN-2.31-1.680.00-1.36-1.98-1.31-0.41-0.39-1.26-1.81-0.46-0.31-0.17-1.31NaN-0.24-0.63-1.80-0.77-0.42-0.77-1.97-0.18NaNQ-0.71-2.03-1.83-0.42-2.14-1.49-1.29NaN-2.540.00-1.40-1.55-0.78-0.45NaN-0.95-0.50-0.800.000.00-0.17-0.83NaN-1.52-0.85-1.85-1.02-1.14-2.15-1.41-1.430.76-2.25-2.99-3.10NaN-0.30-1.93NaNNaN-2.83-1.86-1.85-1.72NaN-0.78-1.36-1.13-0.98NaNNaN-0.32-1.43-0.28-2.95-1.78-2.02NaN-1.84-1.73D-2.02-0.68-1.60-0.33-6.50-2.16-0.53-1.07-2.12-0.47-0.51-1.02NaN-0.88NaN-0.43-1.360.00-2.28-1.91-0.78-1.47-1.72-0.29-1.40-1.79NaN-0.37-1.68-1.79-0.41-0.34-1.81-1.58-0.45-0.44-2.31-2.44-0.30-0.19NaN-0.87-2.040.00-0.41-0.59-0.24-1.36-0.09NaN-0.29-0.67-2.61-0.71-2.23-1.54-0.42-1.360.00-0.52E-0.67-1.53-0.520.00-1.80NaN-1.95NaN-3.02-0.09-0.80-2.00NaN-1.26NaN-1.41-0.11 0.25-0.63-0.86-1.58-1.51NaN-2.12-0.17-0.30NaNNaNNaN-0.47-1.28-0.75NaN-1.32-1.81-1.58-0.72-0.28NaN-1.32-0.26-4.44-1.63-0.24-0.93-1.44-1.52-1.64NaN-1.95NaN-1.28-2.35-1.61-2.49NaN-1.35-1.08-0.17-1.98H-1.92-2.36-3.02-1.42-2.46-1.79-1.63-0.27-1.61-0.26-1.05-0.51-1.33-0.41-0.19-1.98NaN-0.52-0.32-0.79-0.35-1.32-1.36-1.88-1.38-3.07-2.80-1.40-1.30-1.62-1.42-0.16-2.57-0.62-1.76NaN-2.07-2.62-0.79NaN-1.81-1.48-0.24-0.79-1.52-0.67-0.08-0.05-0.26-0.30NaN0.00-1.94-1.06-2.15-2.22-1.89-0.99-0.50-1.65K0.00-2.97-2.23-0.40-1.44NaN-1.63-1.84NaN-0.04NaN-1.71-0.53-2.25NaN-2.590.00-1.75-0.30-0.80-0.67-0.83-0.25-1.620.00NaN-0.42-1.58-1.00-1.49-0.390.07-1.36-2.41NaN-1.40-2.14-2.41-0.30NaN-2.27-1.19-2.17-1.17-1.01-0.81-1.44-0.30NaNNaN-1.48-2.83-3.03-1.52-2.17-3.37NaN-2.83-0.87-1.79R-0.31-1.47-1.61-4.18-1.87-1.97-1.920.00-0.49-0.14-0.97-2.56-1.20-1.62-0.83-1.65-0.20-1.30-0.33-0.80-0.11 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NaN-0.28NaNNaN0.17-0.14-0.27-0.65-0.01-0.16NaNNaN-0.29-0.59-0.48-0.51-0.65-0.34-0.39-0.62-0.71-0.32-0.31-0.10-0.14-0.660.37-0.46-0.18I-1.86-0.56-1.22-0.54-1.51-0.71-0.27NaN-0.05-0.27NaN-1.62-0.86-0.98-2.93-0.34-0.51-0.56-0.08-0.53-0.020.14-0.18NaN-0.51-0.24-0.24-0.39-0.40-0.33-0.41-0.31-0.96-0.07-0.38-0.08-0.10-0.550.16-0.14-0.28L-0.32-0.51-1.38-0.51-1.47-0.69-0.24-0.17-0.990.08-1.53-1.75-0.91-0.47-1.83-1.35-0.35-0.65-0.62-0.76-0.310.00-1.59-0.45-0.59-0.86-0.14-0.48-0.53-0.34-0.33-0.42-0.39-0.20-0.31-0.160.00-0.270.12-0.06-0.18V-1.52-1.21-0.53-0.58-1.96-0.99-0.57-0.790.00-0.72-0.24-0.41-0.10-0.86-0.23-0.89-0.15-0.810.00-0.36-0.65-0.48-1.63NaN-0.21-0.560.01-0.260.00-0.25-0.24-0.69-0.810.00-0.17-0.05-0.14-0.690.12-0.24-0.21A-0.67-1.68-1.13-1.67-1.36-1.64-0.44-1.28-0.43-0.29-0.790.00-0.38-0.120.00-1.31-0.20-0.15-0.44-0.39-0.05-1.46-0.22-1.140.00-0.26-0.07-0.06-0.060.00-0.04-0.45-0.23-0.07-0.020.050.080.020.19-0.04-0.08G-1.66-1.65-0.36-2.53-1.29-0.87-1.36-0.11 -0.41-0.93-0.26-0.69-0.19-0.79-0.50-0.30-0.31-1.03-0.45-0.31-0.48-2.29-1.28-0.570.02-0.41-0.860.00-0.16-0.08-0.13-0.31-0.44-0.160.000.220.05-0.190.300.050.00C-0.97-0.56-1.19-1.23-0.19-0.27-0.65-0.09-1.79-0.45-1.07-0.30NaN-0.83-0.08-1.89-0.69-0.53-0.54-0.59-0.16-2.050.01-0.30-0.22-0.47-0.37-0.30-0.24-0.28-0.260.00-0.49-0.070.50-0.04-0.13-0.680.13-0.24-0.14S-0.37-0.63-0.91-0.65-0.71-0.68-0.71-0.65-1.35-0.13-2.20-0.36-0.87-0.64-0.58-0.46-0.34-0.43-0.90-1.86-0.43-1.61-0.11 -0.570.00-0.07-0.16-0.200.100.15-0.02-0.37-0.17-0.16-0.160.17-0.06-0.200.20-0.20-0.11 T-0.45-2.02-1.06-1.44-2.27-0.970.00-2.12-1.320.00-1.53-0.61-0.63-0.39-0.57-0.84-0.27-0.20-1.24-0.91-0.34-1.800.00-0.98-0.080.000.00-0.160.060.01-0.13-0.42-0.25-0.03-0.220.13-0.11 -0.200.22-0.22-0.24NNaN-1.44-2.03-1.66-0.35-0.30-0.14-0.85-1.230.04-0.06-2.20-0.27-0.48-2.53-1.76-0.610.00-2.47-0.91-0.28-1.70-0.18NaN-0.01-0.05-1.24-0.21-0.26-0.05-0.09-0.210.25-0.25-0.100.21-0.05-0.340.28-0.07-0.14Q-0.11 -1.72-0.42-2.03-1.55-0.84-0.39-1.23NaN-0.01-0.65-1.65-0.26-0.34-1.31-1.03-0.140.22-2.05-0.32-0.12-0.59-1.45-1.950.06-0.21-0.86-0.11 -0.550.010.00-0.34-0.10-0.22-0.090.250.04-0.220.20-0.15-0.23DNaN-0.92-0.07-1.75-0.70-1.00-0.99-1.23-0.24-1.010.00-0.53-0.11 NaN-2.20-1.41-0.27-0.23NaN-0.53-1.00-1.53NaN-0.95-0.37-0.34-1.17-0.11 -0.23-0.010.05-0.18-0.40-0.010.050.380.25-0.380.580.020.14ENaN-1.330.00-1.36NaN-2.00-0.73NaN-1.18-0.48-0.08-2.020.00-0.22-0.41-1.100.00-0.65-0.330.00-0.96-1.47NaNNaN-0.39-0.27-0.83-0.10-0.560.120.12-0.12-0.12-0.11 0.040.290.18-0.260.350.000.07H-1.02-1.81-1.19-1.91-0.26-0.30-1.07NaN-1.92-0.14-0.51-0.25-0.71-0.89-1.55-1.16-0.64-0.11 -1.50-1.24-0.22-1.57NaN-0.32-0.19-0.21-1.26-0.25-0.67-0.20-0.17-0.36-0.44-0.12-0.260.04-0.08-0.450.16-0.35-0.23KNaN-1.19-0.30NaN-1.76-0.03-1.23-0.77NaN-0.28-0.54-1.30-0.220.00-1.95-0.55-0.13-0.44NaN-0.370.03-1.74NaNNaN0.07-0.20-0.53-0.22-0.640.02-0.07-0.40-0.15-0.35-0.160.19-0.15-0.280.070.150.04R-0.71-1.62-1.80-1.23-1.630.00-1.42-0.19NaN-0.27-1.70-1.86-1.07-0.12-2.520.00-0.38-0.55-1.29-0.830.00-0.72-1.660.00-0.060.06-0.96-0.33-0.800.10-0.32-0.400.01-0.39-0.230.00-0.11 -0.35-0.02-0.05-0.10Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged Hydrophobic Aromatic Non-Polar Aliphatic SmallHydrophilic Polar Uncharged Negatively Charged Positively Charged ! 122 Table A 1: Relative fitness for I38V and I22L synonymous codons in the AmiE WT background. Green highlight indicates the codons used for the deep mutational scans presented in this work. Data is from Wrenbeck et al. 24. * !"! Relative Fitness Relative Fitness I38V codon (GTT) -0.17 I122L codon (TTA) -0.89 I38V codon (GTG) -0.43 I122L codon (TTG) -0.69 I38V codon (GTC) -1.17 I122L codon (CTT) -0.46 I38V codon (GTA) -1.26 I122L codon (CTC) -0.31 I38V average -0.35 I122L codon (CTA) -0.54 I122L codon (CTG) -0.69 I122L average -0.53 ! 123 Table A 2: Biophysical analysis of the AmiE variant s. Error values reported are 1 s.d. except in the kinetic analysis where 95% confidence interval is given. ^ >0 represents a less favorable score to the starting wild -type structure (i.e. decreased likelihood folding) * Relative to AmiE WT # Calculated for variant compared to AmiE WT , Expressed via Studiers autoinduction AmiE V ariant WT I122L I38V Rosetta FilterScan score ^ 0.00 0.40 2.02 Relative k cat* 1.00 ± 0.06 1.08 ± 0.06 1.02 ± 0.08 kcat p-value # 1.00 0.57 0.79 Relative K M* 1.00 ± 0.12 0.89 ± 0.15 0.82 ± 0.20 KM p-value # 1.00 0.64 0.32 Relative protein yield ! 1 ± 0.06 0.26 ± 0.04 0.59 ± 0.08 Refolded enzyme velocity normalized to folded enzyme (%) 14.2 ± 2.47 0.114 ± 0.0494 0.064 ± 0.025 0 Growth Rate in M9 (hr-1) - pEDA2 plasmid 0.78 ± 0.01 0.79 ± 0.01 0.78 ± 0.02 Growth Rate in Selection Media (hr-1) - pEDA2 plasmid 0.47 ± 0.02 0.18 ± 0.01 0.11 ± 0.01 Growth Rate in M9 (hr-1) - pAG plasmid 0.79 ± 0.04 0.81 ± 0.03 0.79 ± 0.01 Growth Rate in Selection Media (hr-1) - pAG plasmid 0.72 ± 0.02 0.40 ± 0.03 0.33 ± 0.02 ^ >0 represents a less favorable score to the starting wild -type structure (i .e. de creased likelihood folding ) * Relative to AmiE WT # Calculated for variant compared to AmiE WT ! Expressed via Studiers autoinduction !"!! 124 Table A 3: Thermal shift analysis data and statistics. Summary of thermal melting temperatures (Tm) determined usi ng Boltzmann curve fit of a 2 -state transition. Students t -test p -values are reported for tests comparing AmiE WT with the I38V and I122L variants (N = 6 from two biological replicates of each enzyme; !"indicates 1 s.d. ). #$%&$'(" ")**$+&'," -.'/0'(%$(&.' "1234 "56"1.-4"789$:;0 ")**$+&'," -.'/0'(%$(&. '"1234 "56"1.-4"789$:;0 ")**$+&'," -.'/0'(%$(&.'" 1234 "56"1.-4"789$:;0 ")6&<"=5 ">"?@) "8"A"BCDB"!"EDF "8">E"BGDG"!"EDH "8")6&<"I>HHJ ">"?@) "?@) "A"BCDH"!">DF "EDKH ">E"BGDL"!"> "EDLK ")6&<"IFG# ">"?@) "?@) "A"BCDF"!"EDK "EDH ">E"BGDA"!"EDK "ED>B "! 125 Table A 4: Circular dichroism analysis of thermal denaturation statistics. Summary of apparent thermal melting temperatures (Tm) determined using Boltzmann curve fit of a 2 -state folded -unfolded transition. StudentÕs t-test p -values are reported for tests comparing AmiE WT with the I38V and I122L variants (N = 2 from respective biological replicates of each enzyme). Variant Average T m (oC) Replicate 1 Replicate 2 p-value relative to AmiE WT AmiE WT 69.5 69.5 69.6 - AmiE I122L 67.5 67.9 67.2 0.03 AmiE I38V 67.1 66.9 67.4 0.01 ! 126 Table A 5: DNA sequences of transcriptional elements in plasmids used for deep mutational scans. Differences between sequences are shown in red, the RBS in the 5Õ UTR is underlined. -35 Promoter -10 Promoter 5' UTR pEDA2 Ð AmiE TTCCCG TAATAT TCAGGGAGACCACAACGG TTTCCCTCTACAAATAATT TTGTTTAACTTTCTAGAAA TAATTTTGTTTAACTT TAAGAAGTTTTTATACAT pAG Ð AmiE I38V TTGGCG TACAAT pAG Ð AmiE I122L TTGGCG TACAAT ! 127 Table A 6: Summary of MG1655 rph+ transformants obtained during selection strain mutant library preparation. 5 x 10 4 transformants corresponds to >99% theoretical library coverage. Selection Strain SSM Library Preparation Transformants Obtained AmiE I122L pAG Tile 1 15 x 105 AmiE I122L pAG Tile 2 19x 105 AmiE I122L pAG Tile 3 7.5 x 105 AmiE I122L pAG Tile 4 23 x 105 AmiE I38V pAG Tile 1 6.9 x 105 AmiE I38V pAG Tile 2 5.8 x 105 AmiE I38V pAG Tile 3 7.5 x 105 AmiE I38V pAG Tile 4 7.8 x 105 WT amiE pEDA2 Tile 3 13 x 105 ! 128 Table A 7: Summary of mutational library statistics. !"##$%&'()'*"+%,-' Emily E. Wrenbeck, Laura R. Azouz, Pau l J. Steiner, and Timothy A. Whitehead ''!"#Table S 6: Summary of mutational library statistics. $%$#Screen Growth Enzyme AmiE WT Tile Number 1 2 3 4 Cumulative Residues 1-85 86-170 171-255 256-341 1-341 Population Growth Selected Growth Selected Growth Sele cted Growth Selected Growth Selected Number of mutated codons 85 85 85 86 341 Reference sequencing reads post quality filter 448689 393052 561875 511337 1914953 Selected sequencing reads post quality filter 1454211 3016045 2101231 1691121 8262608 Fold oversampling of codon combinations Average Reference total 81 71.6 101.8 91.6 86.5 Selected total 256.6 544 378.1 292.1 367.7 Reference non -synonymous 50.8 38.3 54.5 55.8 49.9 Selected non -synonymous 79.8 95.7 57.4 95 82.0 Percent of reads with: Average No nonsynonymous mutations 37.6 46.9 46.7 39.4 42.7 One nonsynonymous mutation 60.6 52.2 51.9 59.1 56.0 Multiple nonsynonymous mutation 1.8 0.9 1.4 1.4 1.4 Coverage of possible single nonsynonymous mutations 95.2 93.9 89 94.7 93.2 Screen Growth Enzyme AmiE I122L Tile Number 1 2 3 4 Cumulative Residues 1-85 86-114, 131, 133 -170 171-255 256-341 1-114,131,133 -341 Population Growth Selected Growth Selected Growth Selected Growth Selected Growth Selected Number of mutated codo ns 85 68 85 86 324.0 Reference sequencing reads post quality filter 674718 593530 897746 1214190 3380184 Selected sequencing reads post quality filter 1286349 1002404 1115963 931995 4336711 Fold oversampling of codon combinations Average Reference total 118.7 99 146.8 210 143.6 Selected total 221.8 176.5 198.1 156 188.1 Reference non -synonymous 76.1 61.5 90.1 137 91.2 Selected non -synonymous 71.6 35.8 37.8 63.6 52.2 Reference population percent of reads with: Average No nonsynonymous mutati ons 35.4 35.3 35.2 34 35.0 One nonsynonymous mutation 60.4 55.4 53.7 61.2 57.7 Multiple nonsynonymous mutation 4.3 9.3 11.1 4.8 7.4 Coverage of possible single nonsynonymous mutations 94.5 92.5 93 94.8 93.7 Screen Growth Enzyme AmiE I38V Tile Number 1 2 3 4 Cumulative Residues 1-31, 45 -85 86-170 171-255 256-341 1-31, 45 -341 Population Growth Selected Growth Selected Growth Selected Growth Selected Growth Selected Number of mutated codons 72 85 85 86 328 Reference sequencing reads po st quality filter 550823 500238 525635 490442 2067138 Selected sequencing reads post quality filter 1373021 1060307 991922 1129295 4554545 Fold oversampling of codon combinations Average Reference total 100.7 91.5 96.1 88.6 94.2 Selected total 236.5 189 173.1 185 195.9 Reference non -synonymous 50.3 45.7 48.3 46 47.6 Selected non -synonymous 96.4 42.6 69.8 81 72.5 Percent of reads with: Average No nonsynonymous mutations 50.5 50.5 50.3 48.6 50.0 One nonsynonymous mutation 48.9 48.9 49.2 50.8 49.5 Multiple nonsynonymous mutation 0.6 0.5 0.6 0.6 0.6 Coverage of possible single nonsynonymous mutations 96.7 88.6 87.9 93.9 91.8 $%%# $%&#! 129 Table A 8: Goodness -of-fit test statistics for distribution fittings. For all enzymes the determined p -values indicate a failure to reject the null hypotheses that they can be fit by a general Pareto distribution with a negative shape parameter. All calculations were performed as in Wrenbeck et al. 24. General Pareto dist ribution AmiE WT AmiE I122L AmiE I38V bootstrap test p -value 0.16 0.29 0.10 shape parameter -0.28 -0.33 -0.32 ! 130 Table A 9: Deleterious empirical cumulative distribution function (ECDF) analysis statistics. A table of the two -sample Kolmogorov -Smirnoff test results for comparing the deleterious mutation ECDFs for the three enzymes. Pairing (x -y) K-S Test D p-value Null hypothesis Alternative hypothesis AmiE WT (N) AmiE variant (N) AmiE WT Ð AmiE I38V two sided 0.058 0.0005 is equal to not equal 2428 2472 AmiE WT Ð AmiE I38V greater 0.002 0.99 x not greater than y the CDF of x lies above that of y 2428 2472 AmiE WT Ð AmiE I38V less 0.058 0.0002 x not less than y the CDF of x lies below that of y 2428 2472 AmiE WT Ð AmiE I122L two sided 0.024 0.48 is equal to not equal 2428 2450 AmiE WT Ð AmiE I122L greater 0.010 0.77 x not greater than y the CDF of x lies above that of y 2428 2450 AmiE WT Ð AmiE I122L less 0.024 0.24 x not less than y the CDF of x lies below that of y 2428 2450 ! 131 Table A 10: Inner and outer primers for PCR reactions for Illumina sequencing. Red indicates overhang regions for attaching Illumina adapter primers (inner PCR primers) or overhangs for attaching to inner PCR product (outer PCR primers), black is the overlap region in the gene or the barcode, blue is the Illumina adapter. Inner PC R primers Sequence (5Õ to 3Õ) Fwd_Tile_1 gttcagagttctacagtccgacgatc ttaactttaagaagtttttatacat Fwd_Tile_2 gttcagagttctacagtccgacgatc ggcgaagaaacggaa Fwd_Tile_3 gttcagagttctacagtccgacgatc ctgcgatgacggtaat Fwd_Tile_4 gttcagagttctacagtccgacgatc aagaaatgggcattcaatac Rev_Tile_1 ccttggcacccgagaattcca aagcacggctaaagat Rev_Tile_2 ccttggcacccgagaattcca ctctccaaatttccggata Rev_Tile_3 ccttggcacccgagaattcca cagagacaactgcgc Rev_Tile_4 ccttggcacccgagaattcca tggtggtgctcgag Illumina outer primer adapter aatgatacggcgaccaccgagatctacac gttcagagttctacagtccga Illumina o uter PCR adapters and barcodes RPI37 (AmiE WT unselected, Tile 1) caagcagaagacggcatacgagat ATTCCG gtgactggagttccttggcaccc gagaattcca RPI22 (AmiE WT unselected, Tile 2) caagcagaagacggcatacgagat CGTACG gtgactggagttccttggcaccc gagaattcca RPI39 (AmiE WT unselected, Tile 3) caagcagaagacggcatacgagat GTATAG gtgactggagttccttggcaccc gagaattcca RPI40 (AmiE WT unselected, Tile 4) caagcagaagacggcatacgagat TCTGAG gtgactggagttccttggcaccc gagaattcca RPI41 (AmiE WT selected, Tile 1 replicate 1) caagcagaagacggcatacgagat GTCGTC gtgactggagttccttggcaccc gagaattcca RPI38 (AmiE WT selected, Tile 1 replicate 2) caagcagaagacggcatacgagat AGCTAG gtgactggagttccttggcaccc gagaattcca RPI33 (AmiE WT selected, Tile 2 replicate 1) caagcagaagacggcatacgagat CGCCTG gtgactggagttccttggcaccc gagaattcca RPI34 (AmiE WT selected, Tile 2 replicate 2) caagcagaagacggcatacgagat GCCATG gtgactggagttccttggcaccc gagaattcca RPI43 (AmiE WT selected, Tile 3 replicate 1) caagcagaagacggcatacgagat GCTGTA gtgactggagttccttggcaccc gagaattcca RPI40 (AmiE WT selected, Tile 3 replicate 2) caagcagaagacggcatacgagat TCTGAG gtgactggagttccttggcaccc gagaattcca RPI44 (AmiE WT selected, Tile 4 replicate 1) caagcagaagacggcatacgagat ATTATA gtgactggagttccttggcaccc gagaattcca RPI41 (AmiE WT selected, Tile 4 replicate 2) caagcagaagacggcatacgagat GTCGTC gtgactggagttccttggcaccc gagaattcca RPI20 (AmiE I38V unselected, Tile 1) caagcagaagacggcatacgaga tGGCCAC gtgactggagttccttggcaccc gagaattcca RPI21 (AmiE I38V unselected, Tile 2) caagcagaagacggcatacgagat CGAAAC gtgactggagttccttggcaccc gagaattcca RPI27 (AmiE I38V unselected, Tile 3) caagcagaagacggcatacgagat AGGAATgtgactggagttccttggcaccc gagaattcca ! 132 Table A 10 (contÕd) RPI34 (AmiE I38V unselected, Tile 4) caagcagaagacggcatacgagat GCCATG gtgactggagttccttggcaccc gagaattcca RPI37 (AmiE I38V selected, Tile 1 replicate 1) caagcagaagacggcatacgagat ATTCCG gtgactggagttccttggcaccc gagaattcca RPI38 (AmiE I38V selected, Tile 1 replicate 2) caagcagaagacggcatacgagat AGCTAG gtgactggagttccttggcaccc gagaattcca RPI16 (AmiE I38V selected, Tile 2 replicate 1) caagcagaagacggcatacgagat GGACGG gtgactggagttccttggcaccc gagaattcca RPI14 (AmiE I38V selected, Tile 2 replicate 2) caagcagaagacggcatacgagat GGAACT gtgactggagttccttggcaccc gagaattcca RPI48 (AmiE I38V selected, Tile 3 replicate 1) caagcagaagacggcatacgagat TGCCGA gtgactggagttccttggcaccc gagaattcca RPI23 (AmiE I38V selected, Tile 3 replicate 2 ) caagcagaagacggcatacgagat CCACTC gtgactggagttccttggcaccc gagaattcca RPI32 (AmiE I38V selected, Tile 4 replicate 1) caagcagaagacggcatacgagat TGAGTG gtgactggagttccttggcaccc gagaattcca RPI44 (AmiE I38V selected, Tile 4 replicate 2) caagcagaagacggcatacgagat ATTATA gtgactggagttccttggcaccc gagaattcca RPI19 (AmiE WT control, AmiE I38V selection, unselected, Tile 3) caagcagaagacggcatacgagat TTTCAC gtgactggagttccttggcaccc gagaattcca RPI26 (AmiE WT control, AmiE I38V selection, selected, Tile 3) caagcagaagacggcatacgagat GCTCAT gtgactggagttccttggcaccc gagaattcca RPI33 (AmiE I122L unselected, Tile 1, sequencing runs 1 and 2) caagcagaagacggcatacgagat CGCCTG gtgactggagttccttggcaccc gagaattcca RPI46 (AmiE I122L unselected, Tile 2, sequencing runs 1 and 2) caagcagaagacggcatacgagat TCGGGA gtgactggagttccttggcaccc gagaattcca RPI40 (AmiE I122L unselected, Tile 3, sequencing runs 1and 2) caagcagaagacggcatacgagat TCTGAG gtgactggagttccttggcaccc gagaattcca RPI29 (AmiE I122L unselected, Tile 4, sequencing runs 1 and 2) caagcagaagacggcatacgagat TAGTTG gtgactggagttccttggcaccc gagaattcca RPI17 (AmiE I122L selected, Tile 1 replicate 1) caagcagaagacggcatacgagat CTCTAC gtgactggagttccttggcaccc gagaattcca RPI32 (AmiE I122L selected, Tile 1 replicate 2) caagcagaagacggcatacgagat TGAGTG gtgactggagttccttggcaccc gagaattcca RPI12 (AmiE I122L selected, Tile 2 replicate 1) caagcagaagacggcatacgagat TACAAG gtgactggagttccttggcaccc gagaattcca RPI42 (AmiE I122L selected, Tile 2 replicate 2) caagcagaagacggcatacgagat CGATTA gtgactggagttccttggcaccc gagaattcca RPI25 (AmiE I122L selected, Tile 3 replicate 1) caagcagaagacggcatacgagat ATCAGT gtgactggagttccttggcaccc gagaattcca RPI46 (AmiE I122L selected, Tile 3 replicate 2 ) caagcagaagacggcatacgagat TCGGGA gtgactggagttccttggcaccc gagaa ttcca RPI31 (AmiE I122L selected, Tile 4 replicate 1) caagcagaagacggcatacgagat ATCGTG gtgactggagttccttggcaccc gagaattcca RPI15 (AmiE I122L selected, Tile 4 replicate 2) caagcagaagacggcatacgagat TGACAT gtgactggagttccttggcaccc gagaattcca RPI7 (AmiE WT control, AmiE I122L selection, unselected, Tile 3) caagcagaagacggcatacgagat GATCTG gtgactggagttccttggcaccc gagaattcca RPI10 (AmiE WT control, AmiE I122L selection, selected, Tile 3) caagcagaagacggcatacgagat AAGCTA gtgactggagttccttggcaccc gagaattcca ! 133 Table A 11: Beneficial mutations shared by all enzymes . Location- mutation AmiE WT cumulative normalized fitness metric AmiE WT technical replicate 1 normalized fitness metric AmiE WT technical replicate 2 normalized fitness metric AmiE I122L cumulative normalized fitness metric AmiE I122L technical replicate 1 normalized fitness metric AmiE I122L technical replicate 2 normalized fitness metric AmiE I38V cumulative normalized fitness metric AmiE I38V technical replicate 1 normalized fitness metric AmiE I38V technical replicate 2 normalized fitness metric 9T0.250.240.260.200.190.200.590.600.599Q0.340.340.340.120.11 0.120.450.460.449N0.160.210.11 0.170.140.180.560.560.569I0.280.260.290.270.250.280.490.490.489H0.360.360.360.260.240.280.490.490.509G0.280.250.290.260.250.270.330.310.349D0.260.280.250.410.380.430.600.590.609C0.340.340.340.200.190.210.650.650.659A0.320.310.320.180.190.160.210.220.2193E0.140.140.150.270.260.290.320.320.3293D0.180.170.190.210.200.230.410.400.4293A0.080.070.090.030.030.020.170.160.188T0.200.200.200.140.150.130.520.520.518K0.300.300.300.140.140.140.640.630.648G0.280.280.280.270.260.270.300.310.298A0.100.120.090.050.040.060.550.550.5589S0.090.070.11 0.060.080.030.140.120.1589Q0.150.140.160.280.280.270.250.250.2589N0.150.140.160.160.160.170.300.290.3189E0.300.290.310.360.360.360.730.730.7489D0.230.210.240.300.300.300.570.570.5589A0.180.170.180.310.320.300.470.460.4982Q0.080.070.090.080.080.070.100.090.11 82P0.250.250.260.340.330.340.770.770.7782A0.11 0.100.120.120.130.11 0.190.180.1979V0.040.050.030.100.11 0.090.440.440.4478S0.040.020.050.160.160.160.100.11 0.1074A0.120.130.120.050.050.050.240.220.2570Q0.100.080.11 0.120.11 0.120.180.170.1970K0.130.130.130.200.200.190.390.380.3962T0.090.100.090.090.11 0.080.410.410.4152L0.100.090.11 0.240.230.240.330.330.334S0.160.140.170.060.080.040.410.420.413V0.320.320.330.160.160.150.640.630.643L0.240.240.240.200.200.200.050.030.073I0.270.250.280.230.210.240.610.610.60341D0.130.11 0.140.120.100.130.140.130.14339V0.11 0.100.11 0.140.130.150.120.11 0.13339T0.230.220.230.210.190.220.220.220.21! 134 Table A 11 (contÕd) Location- mutation AmiE WT cumulative normalized fitness metric AmiE WT technical replicate 1 normalized fitness metric AmiE WT technical replicate 2 normalized fitness metric AmiE I122L cumulative normalized fitness metric AmiE I122L technical replicate 1 normalized fitness metric AmiE I122L technical replicate 2 normalized fitness metric AmiE I38V cumulative normalized fitness metric AmiE I38V technical replicate 1 normalized fitness metric AmiE I38V technical replicate 2 normalized fitness metric 339Q0.11 0.100.11 0.190.190.200.200.210.18339P0.230.220.230.210.210.220.270.280.27339N0.300.280.310.280.260.300.280.290.27339M0.100.090.11 0.140.130.140.370.360.38339L0.080.080.080.170.170.170.120.11 0.13339I0.120.130.120.190.180.190.160.150.18339H0.180.160.200.190.170.200.160.150.16339G0.140.120.150.220.210.230.300.300.30339E0.200.180.210.270.270.270.350.360.35339D0.310.290.320.280.280.280.580.580.59339C0.11 0.080.120.090.080.090.130.130.12339A0.200.190.200.130.120.130.190.190.19337E0.270.260.280.11 0.090.120.180.200.16337D0.170.160.170.180.180.180.250.250.24336T0.160.150.160.140.130.150.130.120.13336S0.190.170.190.140.140.140.170.170.16336Q0.240.250.240.200.190.200.250.240.26336P0.250.260.250.210.200.220.250.260.25336N0.280.270.290.230.230.230.210.200.21336K0.210.180.220.220.210.220.190.170.22336H0.070.060.070.080.070.090.040.030.04336G0.240.240.230.180.160.190.220.210.23336E0.310.300.310.230.230.240.290.270.31336D0.330.330.330.270.260.280.380.370.38336A0.070.070.070.050.050.060.050.060.05335D0.090.090.090.060.060.050.050.050.05335C0.410.380.420.320.350.300.500.500.50331E0.210.180.230.100.100.100.120.130.12330S0.020.030.020.070.070.060.150.140.16330E0.160.180.140.130.11 0.160.120.090.14329T0.130.130.130.11 0.130.090.060.060.06329S0.040.050.040.060.060.070.100.090.10322I0.230.210.240.170.160.180.140.140.13312P0.270.250.280.250.270.230.380.380.3828R0.270.270.270.360.360.370.740.740.7428P0.150.160.140.100.100.090.260.250.2728K0.280.260.290.430.420.430.820.810.82279M0.120.100.140.180.150.190.290.300.28279L0.230.210.240.200.200.200.470.460.47! 135 Table A 11 (contÕd) Location- mutation AmiE WT cumulative normalized fitness metric AmiE WT technical replicate 1 normalized fitness metric AmiE WT technical replicate 2 normalized fitness metric AmiE I122L cumulative normalized fitness metric AmiE I122L technical replicate 1 normalized fitness metric AmiE I122L technical replicate 2 normalized fitness metric AmiE I38V cumulative normalized fitness metric AmiE I38V technical replicate 1 normalized fitness metric AmiE I38V technical replicate 2 normalized fitness metric 266H0.160.140.170.060.070.050.190.200.18262A0.140.11 0.150.180.170.180.550.540.55260I0.220.190.230.180.190.160.430.430.43252A0.050.020.070.080.080.090.070.070.06251Y0.160.200.140.150.150.150.240.200.27250P0.210.210.220.210.210.220.200.200.20248S0.150.140.150.200.200.190.11 0.11 0.11 248P0.160.170.160.230.230.230.210.220.21244A0.190.210.190.120.120.120.860.860.86243I0.120.090.130.080.070.090.210.210.22236Y0.270.270.280.210.210.220.640.640.64234M0.330.340.330.190.190.190.880.880.88234C0.280.270.280.140.140.140.770.770.78221T0.040.040.040.100.11 0.100.410.420.41206C0.150.150.140.130.11 0.150.100.11 0.10201M0.370.370.370.310.310.311.041.041.04201L0.160.160.150.170.170.180.850.850.86201I0.200.200.190.200.190.210.710.700.71165C0.270.260.270.390.380.390.960.930.98148T0.120.120.130.140.140.140.120.120.13148S0.080.070.090.100.100.100.070.070.08148N0.190.170.200.250.240.260.400.390.41148K0.100.080.120.11 0.120.100.260.230.30136L0.140.140.150.160.160.160.430.440.43136F0.060.060.060.090.080.090.660.650.6710Q0.170.180.170.190.170.200.130.120.1310A0.230.220.230.170.170.170.530.530.53! 136 Table A 12: Beneficial mutations shared by only AmiE WT and AmiE I122L. Location-mutation AmiE WT cumulative normalized fitness metric AmiE WT technical replicate 1 normalized fitness metric AmiE WT technical replicate 2 normalized fitness metric AmiE I122L cumulative normalized fitness metric AmiE I122L technical replicate 1 normalized fitness metric AmiE I122L technical replicate 2 normalized fitness metric AmiE I38V cumulative normalized fitness metric AmiE I38V technical replicate 1 normalized fitness metric AmiE I38V technical replicate 2 normalized fitness metric 74N0.120.11 0.120.060.060.06-0.07-0.05-0.0882V0.070.070.070.060.060.06-0.09-0.10-0.0888A0.100.090.100.350.350.35-0.09-0.08-0.1092K0.080.070.090.080.070.08-0.13-0.13-0.13112N 0.040.030.050.050.050.05-0.05-0.05-0.06136C0.090.090.090.040.040.05-0.11 -0.16-0.06154D0.050.040.050.050.060.03-0.11 -0.11 -0.10201C0.130.140.130.070.060.09-0.51-0.58-0.45201R0.080.090.070.090.080.10-0.11 -0.10-0.13201T0.200.200.200.130.120.14-0.19-0.19-0.20234Q0.060.070.050.140.150.12-0.28-0.34-0.23248A0.11 0.120.100.140.150.14-0.12-0.15-0.08! 137 Table A 13: Beneficial mutations shared by only AmiE WT and AmiE I38V. Location-mutation AmiE WT cumulative normalized fitness metric AmiE WT technical replicate 1 normalized fitness metric AmiE WT technical replicate 2 normalized fitness metric AmiE I122L cumulative normalized fitness metric AmiE I122L technical replicate 1 normalized fitness metric AmiE I122L technical replicate 2 normalized fitness metric AmiE I38V cumulative normalized fitness metric AmiE I38V technical replicate 1 normalized fitness metric AmiE I38V technical replicate 2 normalized fitness metric 10H0.060.070.05-0.26-0.26-0.260.040.060.039L0.140.130.15-0.14-0.13-0.160.240.250.233Q0.310.310.32-0.07-0.07-0.060.660.650.66339S0.170.160.18-0.03-0.04-0.030.200.200.20237S0.080.040.09-0.19-0.19-0.200.100.130.07! 138 Table A 14: Beneficial mutations shared by only AmiE I38V and AmiE I122L. Location-mutation AmiE WT cumulative normalized fitness metric AmiE WT technical replicate 1 normalized fitness metric AmiE WT technical replicate 2 normalized fitness metric AmiE I122L cumulative normalized fitness metric AmiE I122L technical replicate 1 normalized fitness metric AmiE I122L technical replicate 2 normalized fitness metric AmiE I38V cumulative normalized fitness metric AmiE I38V technical replicate 1 normalized fitness metric AmiE I38V technical replicate 2 normalized fitness metric 4A-0.10-0.13-0.080.080.080.080.550.540.5579C-0.11 -0.11 -0.120.150.140.150.730.720.7369H-0.03-0.04-0.020.040.030.050.030.040.0369R-0.05-0.07-0.030.050.050.050.11 0.11 0.11 ! 139 Table A 15: AmiE WT unique beneficial mutations. Location-mutation AmiE WT cumulative normalized fitness metric AmiE WT technical replicate 1 normalized fitness metric AmiE WT technical replicate 2 normalized fitness metric AmiE I122L cumulative normalized fitness metric AmiE I122L technical replicate 1 normalized fitness metric AmiE I122L technical replicate 2 normalized fitness metric AmiE I38V cumulative normalized fitness metric AmiE I38V technical replicate 1 normalized fitness metric AmiE I38V technical replicate 2 normalized fitness metric 5C0.100.090.11 -0.20-0.18-0.21-0.32-0.32-0.327G0.190.150.21-0.07-0.02-0.12-0.12-0.10-0.1447C0.070.080.07-0.44-0.41-0.46-0.53-0.51-0.5567C0.08-0.050.14-0.08-0.04-0.14-0.13-0.12-0.1472T0.080.090.08-0.62-0.60-0.63-0.60-0.57-0.6374G0.120.11 0.14-0.17-0.15-0.19-0.24-0.25-0.2478H0.11 0.100.12-0.13-0.13-0.13-0.54-0.59-0.4982I0.050.040.06-0.04-0.04-0.03-0.42-0.45-0.39179V0.11 -0.170.17-0.05-0.01-0.10-0.26-0.26-0.27189Y0.08-0.090.12-0.10-0.05-0.15-0.31-0.31-0.30228G0.05-0.100.09-0.16-0.11 -0.23-0.20-0.20-0.21234T0.070.060.08-0.08-0.08-0.09-0.36-0.35-0.37252D0.07-0.250.13-0.21-0.14-0.32-0.54-0.47-0.62277C0.11 -0.470.19-0.49-0.35-0.72-0.30-0.29-0.31291C0.070.100.05-0.50-0.36-0.75-0.40-0.42-0.39297H0.080.11 0.06-0.32-0.42-0.26-0.44-0.46-0.42311C 0.160.200.14-1.17-1.11 -1.22-1.07-1.28-0.94320A0.050.080.03-0.39-0.52-0.32-0.39-0.44-0.34325C0.140.130.14-0.12-0.13-0.12-0.22-0.18-0.27325T0.050.040.06-0.32-0.30-0.33-0.08-0.06-0.09325Y0.160.180.14-0.10-0.01-0.23-0.24-0.22-0.28326H0.220.230.22-0.09-0.10-0.08-0.21-0.27-0.16326Y0.070.090.06-0.27-0.28-0.27-0.51-0.56-0.47336L0.080.070.08-0.04-0.05-0.04-0.16-0.17-0.14341Y0.180.190.17-0.25-0.23-0.27-0.16-0.14-0.18red highlighting indicates samples were dropped from analysis of beneficial mutations due to technical replicate disparities ! 140 Table A 16: AmiE I122L unique beneficial mutations. Location-mutation AmiE I122L cumulative normalized fitness metric AmiE I122L technical replicate 1 normalized fitness metric AmiE I122L technical replicate 2 normalized fitness metric AmiE WT cumulative normalized fitness metric AmiE WT technical replicate 1 normalized fitness metric AmiE WT technical replicate 2 normalized fitness metric AmiE I38V cumulative normalized fitness metric AmiE I38V technical replicate 1 normalized fitness metric AmiE I38V technical replicate 2 normalized fitness metric 47K0.070.070.06-0.07-0.04-0.08-0.07-0.08-0.0747R0.030.030.02-0.07-0.07-0.07-0.13-0.12-0.1458H0.130.010.20-0.24-0.20-0.27-0.17-0.14-0.2269Q0.040.040.05-0.04-0.06-0.03-0.17-0.16-0.1884I0.170.170.16-0.39-0.44-0.35-0.85-0.92-0.7984V0.150.150.14-0.14-0.16-0.13-0.84-0.83-0.85136V0.050.060.04-0.04-0.04-0.04-0.04-0.06-0.03336V0.020.020.03-0.04-0.05-0.04-0.05-0.03-0.08! 141 Table A 17: AmiE I38V unique beneficial mutations. Location-mutation AmiE I38V cumulative normalized fitness metric AmiE I38V technical replicate 1 normalized fitness metric AmiE I38V technical replicate 2 normalized fitness metric AmiE WT cumulative normalized fitness metric AmiE WT technical replicate 1 normalized fitness metric AmiE WT technical replicate 2 normalized fitness metric AmiE I122L cumulative normalized fitness metric AmiE I122L technical replicate 1 normalized fitness metric AmiE I122L technical replicate 2 normalized fitness metric 8V0.300.300.31-0.74-0.83-0.68-0.36-0.39-0.3321A0.040.040.05-0.19-0.21-0.18-0.28-0.29-0.2721P0.380.380.38-0.11 -0.14-0.09-0.10-0.09-0.11 21S0.070.060.08-0.19-0.20-0.19-0.26-0.26-0.2625C0.140.150.14-0.11 -0.12-0.10-0.04-0.03-0.0465V0.240.250.23-0.28-0.31-0.27-0.27-0.25-0.2969Y0.080.080.08-0.12-0.15-0.10-0.27-0.30-0.2472L0.040.030.04-0.35-0.32-0.37-0.43-0.41-0.4575C0.120.130.10-0.39-0.45-0.35-0.19-0.19-0.1875L0.120.11 0.13-0.27-0.27-0.26-0.23-0.23-0.22102C0.11 0.090.13-0.24-0.22-0.25-0.41-0.42-0.40102I0.540.530.54-0.20-0.20-0.20-0.07-0.07-0.07143R0.050.010.08-0.19-0.19-0.20-0.13-0.13-0.13143T0.300.290.30-0.09-0.11 -0.09-0.04-0.04-0.04149M0.400.390.41-0.25-0.27-0.24-0.25-0.23-0.29149R0.060.040.08-0.16-0.16-0.16-0.36-0.32-0.42190T0.370.380.36-0.49-0.43-0.53-0.37-0.42-0.33193P0.530.540.53-0.47-0.48-0.46-0.08-0.08-0.07197C0.300.340.24-1.20-1.10-1.26-0.81-0.95-0.73201N0.250.270.24-0.11 -0.11 -0.11 -0.07-0.06-0.08204N0.260.260.26-0.28-0.35-0.25-0.16-0.13-0.19212C0.510.500.53-0.37-0.35-0.38-0.33-0.34-0.32212K0.070.090.04-0.33-0.56-0.27-0.60-0.46-0.84212T0.400.400.40-0.25-0.10-0.37-0.24-0.23-0.25212V0.190.200.19-0.48-0.41-0.52-0.37-0.37-0.38228A0.150.130.16-0.32-0.31-0.32-0.25-0.23-0.28230C0.250.250.26-0.18-0.21-0.16-0.31-0.31-0.31251N0.470.470.47-0.10-0.13-0.09-0.15-0.16-0.15253Y0.330.330.32-0.49-0.63-0.44-0.39-0.40-0.37254T0.150.160.14-0.42-0.43-0.41-0.43-0.43-0.44287T0.120.130.10-0.17-0.17-0.17-0.21-0.21-0.20288K0.070.080.06-0.07-0.07-0.07-0.04-0.04-0.03317M0.170.180.16-0.41-0.34-0.46-0.23-0.17-0.29325F0.150.190.10-0.16-0.16-0.16-0.15-0.09-0.22326R0.060.040.08-0.24-0.22-0.26-0.23-0.23-0.24333N0.250.220.28-0.30-0.31-0.29-0.20-0.21-0.20340K0.150.160.13-0.12-0.11 -0.13-0.05-0.04-0.05! 142 Table A 18: AmiE WT unique beneficial mutations with synonymous codon fitness disparities. Position Mutation CodonCodon Frequency Reference population count Cumulative selected population count Selected population count - technical replicate 1 Selected population count - technical replicate 2 Cumulative normalized fitness metric Technical replicate 1 normalized fitness metric Technical replicate 2 normalized fitness metric Variance for synonymous codons of normalized fitness metrics - cumulative Variance for synonymous codons of normalized fitness metrics - technical replicate 1 Variance for synonymous codons of normalized fitness metrics - technical replicate 2 Shared-unique beneficial mutation bin 3LCTA 0.041092381105013310.290.290.280.080.090.05Beneficial for all - 95% CI cutoff 3LCTC 0.10211None None None 0.080.090.053LCTG 0.471202-0.27None -0.120.080.090.053LCTT 0.12291094366-0.10-0.12-0.090.080.090.053LTTA 0.140615None None None 0.080.090.053LTTG0.136000None None None 0.080.090.053QCAA 0.341444028167423540.330.330.340.520.321.06WT and I38V shared beneficial - >10% increase in growth rate cutoff 3QCAG 0.6616972-0.69-0.47-1.120.520.321.063VGTA 0.17229083685400.400.390.410.120.060.34Beneficial for all - >10% increase in growth rate cutoff 3VGTC0.2914465790.220.230.220.120.060.343VGTG0.353651-0.27-0.09-0.680.120.060.343VGTT0.286000None None None 0.120.060.345CTGC0.54777993324670.130.130.140.050.060.05WT unique beneficial - 95% CI cutoff 5CTGT0.4618481929-0.19-0.21-0.180.050.060.057GGGA0.134312-0.58-0.67-0.540.240.370.21Unique WT beneficial - >10% increase in growth rate cutoff 7GGGC0.3712117996391160 0.210.170.230.240.370.217GGGG0.159514-0.70-1.01-0.580.240.370.217GGGT0.350101None None None 0.24None 0.218AGCA 0.23111740.150.230.050.420.390.46Beneficial for all - 95% CI cutoff 8AGCC 0.2610211-1.15-1.06-1.240.420.390.468AGCG 0.33414342032310.140.160.120.420.390.468AGCT 0.1878637490.170.170.170.420.390.468KAAA0.742505-0.21None -0.070.13None 0.07Beneficial for all - >10% increase in growth rate cutoff 8KAAG0.26368773765010.310.310.310.13None 0.079LCTA 0.046413-0.63-0.83-0.540.760.280.54WT and I38V shared beneficial - >10% increase in growth rate cutoff 9LCTC 0.14211-0.74-0.67-0.800.760.280.549LCTG 0.4747303-1.92None -1.510.760.280.549LTTA 0.146613175537640.270.260.270.760.280.549LTTG0.1376920490.120.040.170.760.280.5410AGCA 0.23973524149620280.380.380.380.120.120.12Beneficial for all - >10% increase in growth rate cutoff 10AGCC 0.2616010834636200.040.040.040.120.120.1210AGCG 0.3393451511940.390.390.390.120.120.1210AGCT 0.1846733043-0.34-0.35-0.340.120.120.1210HCAC 0.438012465097370.220.210.230.030.030.04WT and I38V shared beneficial - 95% CI cutoff 10HCAT 0.572501206566640-0.04-0.02-0.060.030.030.0447CTGC0.542342325102912960.120.130.120.11 0.140.09WT unique beneficial - 95% CI cutoff 47CTGT0.46801284484-0.34-0.40-0.300.11 0.140.0952LCTA 0.04669053635420.190.180.200.000.000.00Beneficial for all - 95% CI cutoff 52LCTC 0.1857853334520.11 0.11 0.11 0.000.000.0052LCTG 0.47736212583630.090.080.090.000.000.0052LCTT 0.1226194721220.060.030.080.000.000.0052LTTA 0.1496730370.060.070.050.000.000.0052LTTG0.13874911992920.00-0.020.000.000.000.0062TACA 0.17231245866-0.010.01-0.030.010.000.01Beneficial for all - 95% CI cutoff 62TACC 0.4334011712300.170.170.170.010.000.0162TACG 0.2524187841030.070.080.060.010.000.0162TACT 0.19106728390.040.030.040.010.000.0167CTGC0.54464271033240.11 -0.020.171.05None 0.74WT unique beneficial - 95% CI cutoff 67CTGT0.467101-1.34None -1.051.05None 0.7472TACA 0.1710110-1.57-1.06None 0.620.310.42WT unique beneficial - 95% CI cutoff 72TACC 0.4566922993930.170.170.170.620.310.4272TACG 0.25271225567-0.06-0.04-0.070.620.310.4272TACT 0.197321-0.80-0.62-1.050.620.310.4274GGGA0.13132655256399-0.03-0.05-0.020.030.030.03WT unique beneficial - 95% CI cutoff 74GGGC0.3723863551-0.10-0.12-0.100.030.030.0374GGGG0.155810454485970.250.250.250.030.030.0374GGGT0.35568903245660.220.190.240.030.030.0378HCAC 0.4351258101157-0.03-0.05-0.020.030.040.03WT unique beneficial - 95% CI cutoff 78HCAT 0.5732511 2142970.220.220.230.030.040.0379VGTA 0.1766333152181-0.03-0.01-0.040.000.000.00Beneficial for all - 95% CI cutoff 79VGTC0.241273119 1540.030.040.030.000.000.0079VGTG0.35363481591890.120.130.11 0.000.000.0079VGTT0.28543861632230.050.050.050.000.000.0082IATA 0.11 613631412220.01-0.010.020.000.000.00WT unique beneficial - 95% CI cutoff 82IATC 0.3915312795347450.090.080.090.000.000.0082IATT 0.4962337125212-0.01-0.050.010.000.000.0093AGCA 0.236812425347080.070.070.080.000.010.00Beneficial for all - 95% CI cutoff 93AGCC 0.2655651266385-0.03-0.05-0.010.000.010.0093AGCG 0.33439914205710.120.120.130.000.010.0093AGCT 0.186815196558640.120.11 0.120.000.010.00179VGTA 0.1714211-1.43-1.15-1.650.600.280.65WT unique beneficial - 95% CI cutoff 179VGTC0.2226308228927930.16-0.11 0.210.600.280.65179VGTG0.35271147-0.88-0.81-0.920.600.280.65179VGTT0.2816202-1.52None -1.290.600.280.65Position Mutation CodonCodon Frequency Reference population count Cumulative selected population count Selected population count - technical replicate 1 Selected population count - technical replicate 2 Cumulative normalized fitness metric Technical replicate 1 normalized fitness metric Technical replicate 2 normalized fitness metric Variance for synonymous codons of normalized fitness metrics - cumulative Variance for synonymous codons of normalized fitness metrics - technical replicate 1 Variance for synonymous codons of normalized fitness metrics - technical replicate 2 Shared-unique beneficial mutation bin 189YTAC 0.41110 1511 22412870.160.000.2011.16 None 6.06WT unique beneficial - 95% CI cutoff 189YTAT 0.5952101-4.56None -3.2811.16 None 6.06201RAGA0.07564981623360.070.080.060.010.010.01WT and I122L shared beneficial - 95% CI cutoff 201RAGG0.040945None None None 0.010.010.01201RCGA 0.0710310373357020.090.11 0.090.010.010.01201RCGC 0.3620892564-0.10-0.12-0.090.010.010.01201RCGG 0.11 1718067113 0.11 0.150.080.010.010.01201RCGT 0.362204160.090.000.120.010.010.01201TACA 0.171893350101823320.210.210.210.010.010.01WT and I122L shared beneficial - 95% CI cutoff 201TACC 0.40642044None None None 0.010.010.01201TACG 0.2510641945-0.01-0.02-0.010.010.010.01201TACT 0.19343541062480.100.100.100.010.010.01221TACA 0.17110 8162225940.020.000.040.000.000.01Beneficial for all - 95% CI cutoff 221TACC 0.419187541330.090.080.090.000.000.01221TACG 0.25281626498-0.030.03-0.070.000.000.01221TACT 0.19585571773800.080.090.080.000.000.01234QCAA 0.3449325972280.00-0.010.000.010.010.01WT and I122L shared beneficial - 95% CI cutoff 234QCAG 0.66333721242480.120.140.11 0.010.010.01234TACA 0.174014734113 -0.15-0.22-0.120.020.040.02WT unique beneficial - 95% CI cutoff 234TACC 0.42953407100424030.120.120.130.020.040.02234TACG 0.2534912071-0.23-0.33-0.200.020.040.02234TACT 0.194716447117 -0.16-0.18-0.160.020.040.02237SAGC0.252019712-0.55-0.49-0.590.11 0.090.12WT and I38V shared beneficial - 95% CI cutoff 237SAGT0.16153401120281-0.24-0.24-0.240.11 0.090.12237STCA 0.1421311021-0.41-0.39-0.420.11 0.090.12237STCC 0.1510928279203-0.24-0.27-0.230.11 0.090.12237STCG 0.1438872958-0.28-0.25-0.290.11 0.090.12237STCT 0.1752283168021510.430.390.440.11 0.090.12251YTAC 0.41224931863070.260.300.240.040.060.04Beneficial for all - >10% increase in growth rate cutoff 251YTAT 0.592514744103-0.03-0.03-0.030.040.060.04252AGCA 0.233119147144-0.02-0.070.000.010.010.01Beneficial for all - 95% CI cutoff 252AGCC 0.2641380912890.080.020.100.010.010.01252AGCG 0.3311642143-0.03-0.01-0.040.010.010.01252AGCT 0.1818219621570.140.120.140.010.010.01277CTGC0.54115 505-2.30None -1.873.49None 2.60WT unique beneficial - 95% CI cutoff 277CTGT0.465314416013810.34-0.130.413.49None 2.60291CTGC0.5488410142268-0.04-0.04-0.030.010.020.01WT unique beneficial - 95% CI cutoff 291CTGT0.4616114266008260.11 0.150.090.010.020.01297HCAC 0.43117 12725227500.160.190.140.070.090.06WT unique beneficial - 95% CI cutoff 297HCAT 0.576915954105-0.22-0.23-0.210.070.090.06311 CTGC0.547013285767520.270.310.251.272.681.00Unique WT beneficial - >10% increase in growth rate cutoff 311 CTGT0.4649716-1.32-2.00-1.171.272.681.00320AGCA 0.2357993564-0.30-0.30-0.300.300.130.22WT unique beneficial - 95% CI cutoff 320AGCC 0.2674522428-0.59-0.50-0.660.300.130.22320AGCG 0.3315303-1.14None -0.920.300.130.22320AGCT 0.1816819677941173 0.170.200.160.300.130.22325TACA 0.17183021091930.250.250.250.010.020.01WT unique beneficial - 95% CI cutoff 325TACC 0.4121544181363-0.05-0.06-0.040.010.020.01325TACG 0.25109324690.120.050.150.010.020.01325TACT 0.19413561202360.11 0.100.11 0.010.020.01326YTAC 0.411471155 4507050.090.11 0.070.100.080.11 WT unique beneficial - 95% CI cutoff 326YTAT 0.59111679-0.35-0.29-0.400.100.080.11 330SAGC0.2582429145284-0.01-0.020.000.000.000.00Beneficial for all - 95% CI cutoff 330SAGT0.16795191613580.050.010.060.000.000.00330STCA 0.141709473386090.010.010.010.000.000.00330STCC 0.1525113665897770.000.05-0.030.000.000.00330STCG 0.141057922685240.080.070.080.000.000.00330STCT 0.17523671272400.060.060.060.000.000.00336LCTA 0.046223886152-0.08-0.08-0.090.020.020.02WT unique beneficial - 95% CI cutoff 336LCTC 0.1100420157263-0.06-0.05-0.070.020.020.02336LCTG 0.475720149152-0.11 -0.21-0.070.020.020.02336LCTT 0.12148219175914320.220.220.220.020.020.02336LTTA 0.143115347106-0.02-0.06-0.010.020.020.02336LTTG0.13401526290-0.09-0.05-0.11 0.020.020.02337EGAA0.68315411-0.02-0.090.010.050.070.04Beneficial for all - 95% CI cutoff 337EGAG0.32173701262440.300.290.300.050.070.04341YTAC 0.41162721041680.250.260.240.120.130.12Unique WT beneficial - >10% increase in growth rate cutoff 341YTAT 0.59817611-0.24-0.24-0.240.120.130.12! 143 Table A 19: AmiE I122L unique beneficial mutations with synonymous codon fitness disparities. Position Mutation CodonCodon Frequency Reference population count Cumulative selected population count Selected population count - technical Selected population count - technical Cumulative normalized fitness metric Technical replicate 1 normalized fitness metric Technical replicate 2 normalized fitness metric Variance for synonymous codons of normalized fitness metrics - cumulative Variance for synonymous codons of normalized fitness metrics - technical replicate 1 Variance for synonymous codons of normalized fitness metrics - technical replicate 2 Shared-unique beneficial mutation bin 3IATA 0.11 14117 57600.190.190.180.400.370.43Beneficial for all - >10% increase in growth rate cutoff 3IATC 0.39516064960.430.400.460.400.370.433IATT 0.498211-0.76-0.74-0.790.400.370.433LCTA 0.04294382142240.300.300.300.270.140.19Beneficial for all - 95% CI cutoff 3LCTC 0.17532-0.41-0.34-0.490.270.140.193LCTG 0.475101-0.85None -0.610.270.140.193LCTT 0.12249337560.03-0.010.060.270.140.193LTTA 0.141016886820.320.330.310.270.140.193LTTG0.134110-0.76-0.50None 0.270.140.193VGTA 0.171610553520.140.150.130.190.120.11 Beneficial for all - >10% increase in growth rate cutoff 3VGTC0.215525300.520.510.530.190.120.11 3VGTG0.352422-0.13-0.12-0.140.190.120.11 3VGTT0.284220-0.52-0.30None 0.190.120.11 4AGCA 0.232814157840.080.050.11 0.070.070.08I122L and I38V shared beneficial - >95% CI cutoff 4AGCC 0.26453951971980.200.200.190.070.070.084AGCG 0.3319583424-0.030.02-0.080.070.070.084AGCT 0.1832221111-0.42-0.40-0.440.070.070.084SAGC0.25613351711640.100.120.090.010.010.03Beneficial for all - 95% CI cutoff 4SAGT0.1617402614-0.09-0.02-0.190.010.010.034STCA 0.141425178-0.16-0.07-0.290.010.010.034STCC 0.15156933360.060.060.060.010.010.034STCG 0.1474829190.150.190.090.010.010.034STCT 0.1752311120.060.060.060.010.010.038AGCA 0.23178233490.070.040.100.230.170.32Beneficial for all - 95% CI cutoff 8AGCC 0.2638853-0.83-0.72-0.980.230.170.328AGCG 0.331546913183730.060.050.070.230.170.328AGCT 0.1820214901240.240.210.260.230.170.329QCAA 0.3416216961200.280.270.290.11 0.080.14Beneficial for all - 95% CI cutoff 9QCAG 0.6628452619-0.18-0.13-0.240.11 0.080.1410AGCA 0.2312213436417020.240.240.240.120.120.11 Beneficial for all - >10% increase in growth rate cutoff 10AGCC 0.26119 5842693150.080.070.080.120.120.11 10AGCG 0.339915827578250.310.310.310.120.120.11 10AGCT 0.18122763442-0.45-0.47-0.440.120.120.11 47RAGA0.07154423215208-0.05-0.04-0.070.000.000.00I122L unique beneficial - 95% CI cutoff 47RAGG0.041084622242380.050.050.050.000.000.0047RCGA 0.073141132 5815510.010.03-0.010.000.000.0047RCGC 0.3639016768358410.050.060.040.000.000.0047RCGG 0.11 1496072843230.040.030.040.000.000.0047RCGT 0.362308834424410.020.030.010.000.000.0069RAGA0.07102327145182-0.02-0.030.000.000.000.00I122L and I38V shared beneficial - 95% CI cutoff 69RAGG0.04513001351650.120.100.130.000.000.0069RCGA 0.071565892713180.020.010.030.000.000.0069RCGC 0.362038994454540.060.060.050.000.000.0069RCGG 0.11 1245372432940.050.040.060.000.000.0069RCGT 0.361597803714090.080.080.080.000.000.0089SAGC0.25584242401840.150.150.140.010.010.01Beneficial for all - 95% CI cutoff 89SAGT0.165217810969-0.020.01-0.050.010.010.0189STCA 0.1427905436-0.020.00-0.050.010.010.0189STCC 0.15239554410.030.040.010.010.010.0189STCG 0.14129770270.170.220.060.010.010.0189STCT 0.1726885632-0.020.02-0.070.010.010.0193AGCA 0.231286723713010.080.080.070.000.000.00Beneficial for all - 95% CI cutoff 93AGCC 0.26128393215178-0.04-0.04-0.040.000.000.0093AGCG 0.33864102251850.060.060.050.000.000.0093AGCT 0.1876259157102-0.020.01-0.050.000.000.00136VGTA 0.171227503953550.11 0.100.120.000.000.00I122L unique beneficial - 95% CI cutoff 136VGTC0.2169600339261-0.010.00-0.020.000.000.00136VGTG0.35953782221560.020.04-0.010.000.000.00136VGTT0.281065353212140.070.090.040.000.000.00148SAGC0.25693972141830.100.100.100.000.000.00Beneficial for all - 95% CI cutoff 148SAGT0.162313374590.100.100.090.000.000.00148STCA 0.142516982870.130.11 0.150.000.000.00148STCC 0.15533732191540.140.150.120.000.000.00148STCG 0.1438212118 940.090.100.080.000.000.00148STCT 0.17311015249-0.03-0.04-0.010.000.000.00154DGAC0.37492881571310.100.100.100.010.000.01I122L and WT shared beneficial - 95% CI cutoff 154DGAT 0.6355189118 71-0.010.02-0.060.010.000.01244AGCA 0.2330215102113 0.200.190.210.010.010.02Beneficial for all - 95% CI cutoff 244AGCC 0.26704952532420.190.200.190.010.010.02244AGCG 0.33106526390.180.130.210.010.010.02244AGCT 0.1894204110 94-0.05-0.03-0.080.010.010.02336VGTA 0.172333601721880.050.060.040.000.000.00I122L unique beneficial - 95% CI cutoff 336VGTC0.2287341149192-0.01-0.020.000.000.000.00336VGTG0.352333541581960.050.040.050.000.000.00336VGTT0.281902541041500.02-0.010.040.000.000.00! 144 Table A 20: AmiE I38V unique beneficial mutations with synonymous codon fitness disparities. !Position Mutation CodonCodon Frequency Reference population count Cumulative selected population count Selected population count - technical replicate 1 Selected population count - technical replicate 2 Cumulative normalized fitness metric Technical replicate 1 normalized fitness metric Technical replicate 2 normalized fitness metric Variance for synonymous codons of normalized fitness metrics - cumulative Variance for synonymous codons of normalized fitness metrics - technical replicate 1 Variance for synonymous codons of normalized fitness metrics - technical replicate 2 Shared-unique beneficial mutation bin 3IATA 0.11 23193710349030.640.650.640.130.11 0.18Beneficial for all - >10% increase in growth rate cutoff 3IATC 0.39312361620.530.520.540.130.11 0.183IATT 0.4951284-0.030.03-0.140.130.11 0.183LCTA 0.0424210991110.250.230.260.340.220.11 Beneficial for all - 95% CI cutoff 3LCTC 0.112523-0.54-0.61-0.480.340.220.11 3LCTG 0.4711110-1.19-0.85None 0.340.220.11 3LCTT 0.1243753045-0.11 -0.17-0.070.340.220.11 3LTTA 0.14136531340.130.120.150.340.220.11 4AGCA 0.23151150 5945560.630.630.630.190.280.16I38V and I122L shared beneficial - 95% CI cutoff 4AGCC 0.26382513127712360.610.600.610.190.280.164AGCG 0.33164422220.000.000.000.190.280.164AGCT 0.181213310-0.24-0.47-0.130.190.280.168AGCA 0.23179274804470.570.570.580.310.290.33Beneficial for all - 95% CI cutoff 8AGCC 0.26201165-0.45-0.42-0.480.310.290.338AGCG 0.33692591135012410.510.520.510.310.290.338AGCT 0.18718309159150.810.800.810.310.290.338VGTA 0.1744562292270.690.680.690.920.740.62I38V unique beneficial - >10% increase in growth rate cutoff 8VGTC0.216101-1.41None -1.040.920.740.628VGTG0.35261028-0.57-0.92-0.410.920.740.628VGTT0.28714375680.410.410.410.920.740.629LCTA 0.0415303-0.82None -0.560.440.470.44I38V and WT shared beneficial - >10% increase in growth rate cutoff 9LCTC 0.134422-1.06-1.04-1.070.440.470.449LCTG 0.471615411-0.28-0.47-0.180.440.470.449LCTT 0.1211431-0.59-0.45-0.870.440.470.449LTTA 0.14679825054770.350.350.350.440.470.449LTTG0.133231144870.630.660.590.440.470.449QCAA 0.34158904894010.590.600.580.280.330.24Beneficial for all - 95% CI cutoff 9QCAG 0.6620291217-0.16-0.21-0.120.280.330.2410AGCA 0.23578250422240280.720.720.730.290.280.29Beneficial for all - >10% increase in growth rate cutoff 10AGCC 0.2685233113 1200.00-0.010.010.290.280.2910AGCG 0.33312123105910640.610.600.620.290.280.2910AGCT 0.1887532627-0.41-0.42-0.410.290.280.2910HCAC 0.43994912432480.130.130.140.030.020.05I38V and WT shared beneficial - 95% CI cutoff 10HCAT 0.57103185117 68-0.11 -0.05-0.190.030.020.0521AGCA 0.23692731361370.080.080.090.010.010.01I38V unique beneficial - 95% CI cutoff 21AGCC 0.26861587979-0.10-0.10-0.100.010.010.0121AGCG 0.33722551321230.060.060.050.010.010.0121AGCT 0.18632861311550.11 0.090.130.010.010.0121SAGC0.25612711291420.11 0.090.120.000.010.00I38V unique beneficial - 95% CI cutoff 21SAGT0.16632231031200.060.040.080.000.010.0021STCA 0.14692481271210.060.060.060.000.010.0021STCC 0.158422493131-0.01-0.060.030.000.010.0021STCG 0.1459198951030.040.030.060.000.010.0021STCT 0.1729190106840.190.210.170.000.010.0069RAGA0.07581398356-0.030.01-0.090.020.020.02I38V and I122L shared beneficial - 95% CI cutoff 69RAGG0.04507453693760.350.350.360.020.020.0269RCGA 0.07102270139131-0.01-0.01-0.020.020.020.0269RCGC 0.361235382772610.100.11 0.100.020.020.0269RCGG 0.11 923491681810.070.060.080.020.020.0269RCGT 0.361214602312290.070.070.070.020.020.0272LCTA 0.043416181800.120.120.120.000.000.00I38V unique beneficial - 95% CI cutoff 72LCTC 0.1873071551520.060.060.060.000.000.0072LCTG 0.471013191361830.03-0.010.060.000.000.0072LCTT 0.1263212114 980.050.060.030.000.000.0072LTTA 0.14401075057-0.01-0.030.010.000.000.0072LTTG0.1385227115 112 -0.01-0.01-0.010.000.000.0093AGCA 0.23651828399-0.03-0.070.010.050.050.06Beneficial for all - 95% CI cutoff 93AGCC 0.2637773938-0.10-0.12-0.090.050.050.0693AGCG 0.33418204403800.380.380.390.050.050.0693AGCT 0.1822452718-0.11 -0.08-0.150.050.050.06102CTGC0.54312101041060.170.150.190.320.320.32I38V unique beneficial - 95% CI cutoff 102CTGT0.4611422-0.63-0.65-0.620.320.320.32143RAGA0.0721321715-0.18-0.18-0.180.020.010.03I38V unique beneficial - 95% CI cutoff 143RAGG0.0440735023-0.14-0.07-0.240.020.010.03143RCGA 0.0742563125-0.22-0.21-0.230.020.010.03143RCGC 0.3615410554536020.170.120.220.020.010.03143RCGG 0.11 59112 5755-0.13-0.14-0.11 0.020.010.03143RCGT 0.3653904941-0.16-0.15-0.160.020.010.03Position Mutation CodonCodon Frequency Reference population count Cumulative selected population count Selected population count - technical replicate 1 Selected population count - technical replicate 2 Cumulative normalized fitness metric Technical replicate 1 normalized fitness metric Technical replicate 2 normalized fitness metric Variance for synonymous codons of normalized fitness metrics - cumulative Variance for synonymous codons of normalized fitness metrics - technical replicate 1 Variance for synonymous codons of normalized fitness metrics - technical replicate 2 Shared-unique beneficial mutation bin 143TACA 0.1730673829-0.09-0.07-0.100.050.050.06I38V unique beneficial - >10% increase in growth rate cutoff 143TACC 0.4902119 1113 10060.410.400.420.050.050.06143TACG 0.254214173680.010.010.020.050.050.06143TACT 0.1926703931-0.04-0.03-0.050.050.050.06149RAGA0.0711303-0.74None -0.470.200.280.18I38V unique beneficial - 95% CI cutoff 149RAGG0.0415312-0.87-1.07-0.730.200.280.18149RCGA 0.07231275-0.51-0.47-0.550.200.280.18149RCGC 0.3619936-0.54-0.70-0.430.200.280.18149RCGG 0.11 705992983010.220.200.240.200.280.18149RCGT 0.3615211-1.06-1.07-1.040.200.280.18197CTGC0.5413312-0.70-0.87-0.600.650.900.48I38V unique beneficial - >10% increase in growth rate cutoff 197CTGT0.4612199124750.440.470.390.650.900.48201NAAC0.51851749183-0.010.00-0.020.060.060.06I38V unique beneficial - >10% increase in growth rate cutoff 201NAAT 0.49118 1150 6175330.330.350.320.060.060.06228AGCA 0.2336743-0.77-0.71-0.850.290.300.31I38V unique beneficial - >10% increase in growth rate cutoff 228AGCC 0.26363931862070.360.340.370.290.300.31228AGCG 0.3318422-0.72-0.71-0.730.290.300.31228AGCT 0.1812312-0.67-0.83-0.570.290.300.31237SAGC0.2553472423-0.24-0.23-0.250.060.060.05I38V and WT shared beneficial - 95% CI cutoff 237SAGT0.16841115259-0.13-0.14-0.11 0.060.060.05237STCA 0.1414271215-0.03-0.060.000.060.060.05237STCC 0.15445593382210.390.420.340.060.060.05237STCG 0.1430492425-0.07-0.07-0.070.060.060.05237STCT 0.17181477-0.28-0.28-0.280.060.060.05251YTAC 0.41373771592180.340.310.370.060.060.06Beneficial for all - >10% increase in growth rate cutoff 251YTAT 0.5938803644-0.01-0.030.020.060.060.06252AGCA 0.23541045549-0.03-0.01-0.040.040.040.05Beneficial for all - 95% CI cutoff 252AGCC 0.2613319494100-0.10-0.11 -0.090.040.040.05252AGCG 0.3352744034-0.11 -0.09-0.130.040.040.05252AGCT 0.18434232162070.340.340.330.040.040.05287TACA 0.17242391361030.310.330.290.060.050.07I38V unique beneficial - 95% CI cutoff 287TACC 0.4257232400.040.010.070.060.050.07287TACG 0.2534372116-0.21-0.18-0.250.060.050.07287TACT 0.196743-0.19-0.16-0.230.060.050.07288KAAA0.741688554643910.170.180.160.060.050.06I38V unique beneficial - 95% CI cutoff 288KAAG0.26164214115 99-0.16-0.15-0.180.060.050.06325FTTC0.42542881791090.180.220.130.180.470.08I38V unique beneficial - >10% increase in growth rate cutoff 325FTTT0.589514-0.42-0.75-0.270.180.470.08326RAGA0.0732431924-0.15-0.19-0.120.090.100.10I38V unique beneficial - 95% CI cutoff 326RAGG0.0415963-0.39-0.31-0.530.090.100.10326RCGA 0.0734251312-0.33-0.32-0.340.090.100.10326RCGC 0.36534812282530.290.270.310.090.100.10326RCGG 0.11 241358-0.43-0.51-0.360.090.100.10326RCGT 0.36271046-0.56-0.64-0.490.090.100.10329SAGC0.25721566888-0.03-0.070.010.020.030.02Beneficial for all - 95% CI cutoff 329SAGT0.1683515200.140.090.180.020.030.02329STCA 0.14359747500.030.020.050.020.030.02329STCC 0.15113 258105153-0.02-0.070.030.020.030.02329STCG 0.14481055847-0.03-0.01-0.050.020.030.02329STCT 0.17435052822230.340.350.330.020.030.02329TACA 0.174314375680.080.080.070.000.000.01Beneficial for all - 95% CI cutoff 329TACC 0.4922951511440.070.060.070.000.000.01329TACG 0.2525512922-0.04-0.02-0.070.000.000.01329TACT 0.1931110 52580.090.070.11 0.000.000.01330SAGC0.251263761801960.050.030.070.030.020.03Beneficial for all - 95% CI cutoff 330SAGT0.16811729775-0.03-0.01-0.060.030.020.03330STCA 0.141381129 5865430.270.270.270.030.020.03330STCC 0.15208453213240-0.03-0.05-0.010.030.020.03330STCG 0.141011182 5466360.340.320.370.030.020.03330STCT 0.171303411801610.020.020.010.030.020.03333NAAC0.51263011331680.340.310.370.180.160.19I38V unique beneficial - >10% increase in growth rate cutoff 333NAAT 0.49171688-0.25-0.26-0.250.180.160.19336AGCA 0.231304222222000.070.070.070.000.010.00Beneficial for all - 95% CI cutoff 336AGCC 0.261505793172620.11 0.120.100.000.010.00336AGCG 0.339519410292-0.04-0.04-0.050.000.010.00336AGCT 0.18102239113 126-0.01-0.030.010.000.010.00340KAAA0.741498314653660.190.210.170.060.060.06I38V unique beneficial - >10% increase in growth rate cutoff 340KAAG0.2648633429-0.16-0.14-0.180.060.060.06! 145 APPENDIX B Chapter 4 supporting information ! 146 Note B 1: DNA sequences of !X174 viral capsid protein gene fragments. Sequences provided span from the 5Õ EcoRV to the 3Õ BamHI site (bolded sequences). Mutagenic regions are highlighted in pink, BsmBI sites are highlighted in green. >Fragment_F1 GATATC TGCAGAATTCGCCCTT CGTCTC ATTCAAACGGCCTGTCTCATCATGGAAGG CGCTGAATTTACGGAAAACATTATTAATGGCGTCGAGCGTCCGGTTAAAGCCGCTGA ATTGTTCGCGTTTACCTTGCGTGTACGCGCAGGAAACACTGACGTTCTTACTGACGC AGAAGAAAACGTGCGTCAAAAATTACGTGCAGAAGGAGTGATGTAATGTCTAAAGG TAAAAAACGTTCTGGCGCTCGCCCTGGTCGTCCGC AGCCGTTGCGAGGTACTAAAGG CAAGCGTAAAGGCGCTCGTCTTTGGTATGTAGGTGGTCAACAATTTTAATTGCAGGG GCTTCGGCCCCTTACTTGAGGATAAATT ATGTCTAATATTCAAACTGGCGCCGAGCG TATGCCGCATGACCTTTCCCATCTTGGCTTCCTTGCTGGTCAGATTGGTCGTCTTATT ACCATTTCAACTACTCCGGTTATCGCTGGCGACTCCTTCGAGATGGACGCCGTTGGC GCTCT CCGTCTTTCTCCATTGCGTCGTGGCCTTGCTATTGACTCTACTGTAGACATTTT TACTTTTTATGTCCCTCATCGTCACGTTTATGGTGAACAGTGGATTAAGTTCATGAAG GATGGTGTTAATGCCACTCCTCTC CCGACTGT GAGACG AAGGGCGAATTCCAGCACA CTGGGCGGCCGTTACTAGT GGATCC >Fragment_F2 GATATC TGCAGAATTCGCCCTT CGTCTC GACTGTT AACACTACTGGT TATATTGACC ATGCCGCTTTTCTTGGCACGATTAACCCTGATACCAATAAAATCCCTAAGCATTTGTT TCAGGGTTATTTGAATATCTATAACAACTATTTTAAAGCGCCGTGGATGCCTGACCG TACCGAGGCTAACCCTAATGAGCTTAATCAAGATGATGCTCGTTATGGTTTCCGTTG CTGCCATCTCAAAAACATTTGGACTGCTCCGCTTCCTCCTGAGACTGAGCTTTCTCGC CAAATGACGACTTCTA CCACATCTATTGACATTATGGGTCTGCAAGCTGCTTATGCT AATTTGCATACTGACCAAGAACGTGATTACTTCATGCAGCGTTACCGTGATGTTATT TCTTCATTTGGAGGTAAAACCTCTTATGACGCTGACAACCGTCCTTTACTTGTCATGC GCTCTAAT CTCTGGG GAGACG AAGGGCGAATTCCAGCACACTGGCGGCCGTTACTA GTGGATCC >Fragment_F3 GATATC TGCAGAATTCGCCCT TCGTCTC TCTGGGCA TCTGGCTATGATGTTGATGGA ACTGACCAAACGTCGTTAGGCCAGTTTTCTGGTCGTGTTCAACAGACCTATAAACAT TCTGTGCCGCGTTTCTTTGTTCCTGAGCATGGCACTATGTTTACTCTTGCGCTTGTTCG TTTTCCGCCTACTGCGACTAAAGAGATTCAGTACCTTAACGCTAAAGGTGCTTTGAC TTATACCGATATTGCTGGCGACCCTGTTTTGTATGGCAACTTGCCGC CGCGTGAAATT TCTATGAAGGATGTTTTCCGTTCTGGTGATTCGTCTAAGAAGTTTAAGATTGCTGAG GGTCAGTGGTATCGTTATGCGCCTTCGTATGTTTCTCCTGCTTATCACCTTCTTGAAG GCTTCCCATTCATTCAGGAACCGCCTTCTGGTGATTTGCAAGAACGCGTACTTATTCG CCACCATGATTATGACCAGTGTTTCCAGTCCGTTCAGTTGTTGCAGTGGAATAGTCA GGTTAAATTTAATGTGACCGTTTATCGCAATCTGCCGACCACTCGCGATTCAATCAT GACTTCG TGATAAAAGATTGAGTGTGANNNNNNNNNNNNGGTTATAACGCCGAAGC GGTAAAAATTTTAATTTTTGCCGCTGAGGGGTTGACCAAGCGAAGC GAGACG AANGGCGAATTCCAGCACACTGGCGGCCGTTACTAGT GGATCC ! 147 >Fragment_G1 GATATC TGCAGAATTCGCCCTT CGTCTC CGAAGCGCGGTAGGTTTTCT GCTTAGGAG TTTAATCATG TTTCAGACTTTTATTTCTCGCCATAATTCAAACTTTTTTTCTGATAAGC TGGTTCTCACTTCTGTTACTCCAGCTTCTTCGGCACCTGTTTTACAGACACCTAAAGC TACATCGTCAACGTTATATTTTGATAGTTTGACGGTTAATGCTGGTAATGGTGGTTTT CTTCATTGCATTCAGATGGATACATCTGTCAACGCCGCTAATCAGGTTGTTTCTGTTG GTGCTGATATTGCT TTTGATGCCGACCCTAAATTTTTTGCCTGTTTGGTTCGCTTTGA GTCTTCTTCGGTTCCGACTACCCTCCCGACTGCCTATGATGTTTATCCTTTGAATGGT CGCCATGATGGTGGTTATTATACCGTCAAGGACTGTGTGACTATTGACGTCCTTCCC CGTACG GAGACG AAGGGCGAATTCCAGCACACTGGCGGCCGTTACTAGT GGATCC >Fragment_G2 GATATC TGCAGAATTCGCCCTT CGTCT CCGTACG CCGGGCAATAATGTTTATGTTGG TTTCATGGTTTGGTCTAACTTTACCGCTACTAAATGCCGCGGATTGGTTTCGCTGAAT CAGGTTATTAAAGAGATTATTTGTCTCCAGCCACTTAAG TGAGGTGATTTATGTTTGGTGCTATTGCTGGCGGTATTGCTTCTGCTCTTGCTGGTGGCGCCATGTCTAAATTGTT TGGAGGCGGTCAAAAAGCCGCCTCCGGTGGCATTCAAGGTGATGTGCTTGCTA CCGATAACAATACTGTAGGCATGGGTGATGCTGGTATTAAATCTGCCATTCAAGGCTCTA ATGTTCCTAACCCTGATGAGGCCGCCCCTAGTTTTGTTTCTGGTGCTATGGCTAAAGC TGGTAAAGGACTTCTTGAAGGTACGTTGCAGGCTGGCACTTCTGCCGTTTCTGATAA GTTGCTTGATTTGGTTGGACTTGGTGGCAAGTCTGCCGCTGATAAAGGAAAGGATAC TCG GAGACG AAGGGCGAATTCCAG CACACTGGCGGCCGTTACTAGT GGATCC ***********************! 148 Note B 2: Amino acid sequences of !X174 mutagenized viral capsid protein gene fragments. Only mutagenized regions of gene fragments shown. >Fragment_F1 MSNIQTGAERMPHDLSHLGFLAGQIGRLITISTTPVIAGDSFEMDAVGALRLSPLRRGLAI DSTVDIFTFYVPHRHVYGEQWIKFMKDGVNATPL >Fragment_F2 NTTGYIDHAAFLGTINPDTNKIPKHLFQGYLNIYNNYFKAPWMPDRTEANPNELNQDDA RYGFRCCHLKNIWTAPLPPETELSRQMTTSTTSIDIMGLQAAYANLHTDQERDYFMQRY RDVISSFGGKTSYDADNRPLLVMRSN >Fragment_F3 SGYDVDGTDQTSLGQFSGRVQQTYKHSVPRFFVPEHGTMFTLALVRFPPTATKEIQYLN AKGALTYTDIAGDPVLYGNLPPREISMKDVFRSGDSSKKFKIAEGQWYRYAPSYVSPAY HLLEGFPFIQEPPSGDLQERVLIRHHDYDQCFQSVQLLQWNSQVKFNVTVYRNLPTTRD SIMTS >Fragment_G1 FQTFISRHNSNFFSDKLVLTSVTPASSAPVLQTPKATSSTLYFDSLTVNAGNGGFLHCIQM DTSVNAANQVVSVGADIAFDADPKFFACLVRFESSSVPTTLPTAYDVYPLNGRHDGGY YTVKDCVTIDVLP >Fragment_G2 PGNNVYVGFMVWSNFTATKCRGLVSLNQVIKEIICLQPLK !!!!!!!!!!!!!!!!**! 149 *Figure B 1: Introduction of nicking sites into shuttle vectors containing viral genes. Verification of the introduction of the BbvCI nicking site into the shuttle vector containing the viral genes is shown by the generation of ssDNA as in the NSM protocol. Samples we re run on a 1% agarose gel with SYBRª Safe DNA gel stain (Invitrogen) added before casting, the ladder used is the 1 kb DNA ladder from GoldBio. ************!"#$"##$%"#$&###$&"##$!###$'()*+,-.,)$ /01-2*)$345$ --345$ 6&$ 6!$ 67$ 8&$8!$6&$ 6!$ 67$ 8&$8!$!"#$"##$%"#$&###$&"##$!###$&$9:$01)),;$ &$9:$01)),;$ ! 150 ***Figure B 2: F1 heatmap of counts. **********************F1 heatmap of counts Location 123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960Mutation MSNIQTGAERMPHDLSHLGFLAGQIGRLITISTTPVIAGDSFEMDAVGALRLSPLRRGLA*7674673573403803421521902421583024034288811189 18795976207187997133673239144217128729651314029629254418720346222023121410022136570029452630770644415285119 424444226318566213114 337100F61054730442123726482166148132211 44045163310761308630646533-703189635254147544181275101330173706214143654292359190145276-67525650036686650414716621584264624332371235910966262W9195443884883252691592011932693453846741006104813956975197797664320570597312858894176119 28522421716916930516020915995158240639409409429705419162117 24344846226221663625218041871Y654658467355203314112 325182120261461511 72810961705763607640799752285410318022856532848390277227654238180608261326282112 193477557288572286668395114 94115 4393862083128062889034593P911 827341566420586130318176215295-4999631331128913855315721215123347505396100284104164149177334277184-26613113655101256245604467593417651616146229240471419-18654328097498109M-424511 570254400143267253247-550639946149715491101 5567121053422574265117 863051062158845722448416020638112633517281189258587-55640381748523216023341036135431855533921754393I924424447-2884707729824617341550237768515471272647655554888115 2787935-173368128-186-191637300164705-395224742493145275086224531092526177184229652607360257887378112 67265L14934894374566504961613252482405257485441029-10101079-6961199 -27253112 18387318-223104281237270116 39438616916812680178275482372463280702542136-155-326339-811 348123-62V1180 3743474713313872753293891393914774769511115 122169868771484861294707311124738379234122364231438164136-199493252152206272655437545529-518224117 252473382370262549253184447187A1050646356383440610190-59618440475542710031058140974955877495348-82539519029583180289257263343208192353119 -219195177248501261480-777622-113 221501430399157428294182438-G810583445370302344-467355238309880287128810721584992665-92567368-5652-19675110 170310177373148164260142154-1591502428792941037367713-9969171552444172153454263-28088C1000569270397315394199411 19845335687029910941128 1517565679616847762358531126123439110 15318529526242514416738022331018212629737279527967746571742213113426156954039422275839222449673S1040-415462427498241547319300430165452112981425-741584774840533891384416112632881180221285-520274289307270452306170-535686338738549726521146118 178598-46522973331919137490T827720624639361-12241822916031381027291313161125 600472727571702637358222138352140212-311 203--174295208386141188277302684320612611 748445199100140903581391223496237150309155N733388-411 32151211129922520731073035192595010596568526777527227152501431224241242182183172073911762012812172671312511934396323097714838615281569314989451535923848826910742469Q986370421533-38616936034826438274078387010301193 1151 7296759376729064-149131321136182130278197418130252299100233798015622065835745926995337918850126380369235233542227114 35662D762555531497285685236743303283369516455-81617747405769567946229813435121311 572162264109285249508191143373148421438-160279694357-38393847217920916757255036225270633617728092E707654349346306367227220-2162608035361141 894136857588673765836241665291179550117 213120321234353166170254166338281194230296-28552827389145525814513350552033727940935392301109H746596539323589409147336183409377636-76910791583-964623824111239118 83160172396281252132256212523156256396484225170129141321651399540525934383148115 2684063412922448314227240063K70629846128441840914835736021537343573181912441435544711 72684645217726510015647152202124265231410153165289187271224148297436742247516344873580186117 143561508372267403359201705121R1149 713277363400460146356186-34510054431177 10871616704631630992932407499150129-111177280266174409151250342155242125140207227616366678466786468109142-403317338162--116 42163F1 heatmap of counts Location 6162636465666768697071727374757677787980818283848586878889909192939495Mutation IDSTVDIFTFYVPHRHVYGEQWIKFMKDGVNATPL*1741571211901954302192068465879101564559106336119 55816344535225124155245139224124266208159165297F266141235235247362332-52-5122561403349118 10916438368113 311 188-50173115 158891219144183183313W107172101131193415151172825712192667273487408424094-28426618968256113 20294175184110 51207Y1391252241491943802602468152-2310261338196-198357101873342592694777821145152207185187137269P1551769622320448717113592382317-17857778633782451331502732811774217788108102188178121-259M181129771831993914401749939231746130332913749141277101100629278178-302134242125229172193150255I-154215323297408-25016982407749113 44371685519243763126-173272205249159176143266141268151314L184131100214208412172233791001417145213766212048110 20516722028024835393222110 203139197180122191-V242221155134-523244205774619-331034124-9915733312420736227547385222165398-240633180161289A18220721519020546522224428232187691118 43411075214027013712125827414756217119 193160239-873376230G118 266118 1542151133 23125610238238741110 2550110 11-2768319719721718249216234-154194571148162191C1402362122242531011 2003138441733553841075213095368374115 19222524259343152141334146209265167165269S188272-24122490930521716482352110592412963291512346620751025727149221203298102603323189201244T214232105-226841212159-52193620015221261065310441310897422232165902211061288930398-419254N203273118 290311 106428720113341252488174206374741524309014536432417627407291172125-356264236249Q10512183162179412211 179137282921121223564310658126301-114 2702211547229813116791242307179190351D187-132152361-252227130225497115 1884898157733007427817133225923741199-311 168328332234181217E2602401281603277152581656630212351120353312039108-6918022231722836337938210117 267340192120274H1441601382042871147 3553402051224517207-148-6094160338142130323116 15042191151254112 1712622214831383K23416713619925243724716572241696512229208443116 51478190252-220561-1261731292000216197144275R15424613921319376517614387462925133208-104936214222925423123124420860223194189153167243161198367050100250500!"#$%&' ! 151 *************Figure B 3: F2 tile 1 heatmap of counts. *********************F2 Tile 1 heatmap of counts Location 99100101102103104105106107108109110 111112 113 114 115 116 117 118 119 120121122123124125126127128129130131132133134135136137138139140141142143144145146147148149150151152153154155156157Mutation NTTGYIDHAAFLGTINPDTNKIPKHLFQGYLNIYNNYFKAPWMPDRTEANPNELNQDDA*175178160292072055962516759432161227826815359261209391645134331482012153805923454482401572677783161895074173277534484301678441201168200455987352F2151471994220364241062123-12525130115 3622212846259884910321531-225844890811706289150-122685728113 7069200257410435319571428146234187256899567W176199118 571442943781776196331512772258155542468751176412482610227579927517294641841701437658-1324572204265330442259525373226136196264121112 84Y20320318942-2569142222048123231302713027219661349117 392033641755517-33211 60-79111-213137656923120696420130247449135357844618218729227128426847P220220497312153144365381650178351793664-127121239110 30-603838302597952846116075118 12815714888-19130-80263342418526299-332206234178411 909085M1782001573416637408616135399232865958286119 71270165118 22742050383167763142116 18077931511761741057134-54772382873525033566324572452002412959810174I20522224630206-396827166212830140-110 314179129373128-22431319492286320187-1119314012419616574622726045721853854265324085645441743162852128510462L2132271342817838371542415119 -261493766310116 612599041593662-108421682-6392157811001742661341062337916513371286314276428331675391246-184437112 103110 V178216141771427191882546062866217082672701597225710793145520327575069531201381947575167199129209853120137125232287396490339666369318228227261147172440A182283272741411495112 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462175503536-23734712180151968614721516795126251286371227343332492334515376227183233-1298081D23023622110017345-110 46426212130212736156329-85363121413239441738285782330474240157113 16715217575672210076-241262526598385537477289162308342--7467E1632171565514939112 8636295910638162485425525467279247421011117262613405124122881878987149134363755525112 30146220277-368343649411 -18523839824827587H20621222450311 2852-1920461283117433763602057030188394233-19304022112 291486927475116 198185173697129129112 77381303408476349433437221228208450142116 77K21619017332159156658132548133371804013129115271344-658-23102617196425210861598386152136-666431176397419230354642641062657222319340339510310880R18724227050206197312727284213752199361023041601042421397528775928171432763459196135103135160209184587610713090111-275473485311 642311 201317207478108112 80050100250500!"#$%&' ! 152 ********Figure B 4: F2 tile 2 heatmap of counts. **********F2 Tile 2 heatmap of counts Location 158159160161162163164165166167168169170171172173174175176177178179180181182183184185186187188189190191192193194195196197198199200201202203204205206207208209210211 212213214215216217Mutation RYGFRCCHLKNIWTAPLPPETELSRQMTTSTTSIDIMGLQAAYANLHTDQERDYFMQRYR*1256317334023942447751334340460419699422629119480178271451250354657834280640670590336845398281421226316490238652292915102836431855560851756791240040212138913012670787384279F113 54094-18039237849132430745827568019632414777399246334265318707810292389609474376935313311 60929924657720580219757719926583310577507700402675311 344115 317-121141114 301249W19130692629260389421495315312585188-134314185652134336441166312598712262430670572314950441266569245343502224170216855624423911129555968159958861235439814626015493233122279358Y97-134389206603107363622429148223879922840320785483226384275263679862238464590509381836373263571233280609234342161011022-973235171430693571701314406213-2418015675-344P199338166428255447407421711 297760179753227406-927--43429829197687837755874266154598655537862824535948223885658677436217793265638708721522626371560117 30816198245160292359M122401109511 200389464500290336481196759191363190795151307396241292696776268553-955552855548337565240368613-9737085613011874728759958757253058037944914714159-17590282306I12245183433192350417419279396568-757300341138811 128290326379282844982285503926516400759575439523-326-559892428684233258563643565628614585852723681483418525510277262332L214355135728271399438566-3215761891079191355442-173494382211 341-80434661483367144582944137048724130455842076-19492255167120261-712686515687362496129313314160352176259390V138367198415237393359550309327522296693149382165708115 264511 18540953678834645389248938193647835456730248459037931534156124307182354256535602668732393585362249289191270257140256329A153323185482229456349569341263498252779444-26462315425743142035388483035146973590350477863345360724448145223820527469--219-308536545877629645524397192360129151205128229360G117 353-403286429386439372273610189785167372148578103369571255509538890318459691600380859416313499226796559213-28480101309197170292503526694920616495464246310165131168152285429C264529124490625--507329277678177853206415147855893773602762976828006813997695074049054523596692615655312322792739886259287103304551652682744682380760143537204113 183293610644S206411 233563296455468434385355691236899318388351728210454385412382651-426503714668530-591514-27160470031924626155112 388220148444650578724768624393559154178337112 161128326415T128358114 392273459530528256368876303763-556258713128370365-279665937340484895--819--5683494476333267519887138481210230310444535-7375963344231533417326615384335361N138335117 318237385463538311 424-255889263377208824160366365377369689848338581788513417765569343490269641713242681861058922121482-4395626597597033494742714023212217069343339Q18836286422247375352519336395624193842196333296592167320447282400684874385-64460841999453239955525440350326460400-5923520878271569801625628-426532140289159133-132256420D147408227341242375345531265353699198751236415245656130279468345363616996294499727609446810392287462280-62427833528276158288263200425508605753-692542381-33117299214100304365E117 314135367216316419484238519564202852151398176523100309-266-7298253586126636474049034543255911995006372401212201278626127090311 593509663893702-38222935520512421270337364H31660296302520396323-491301657200327195184261752147382361334345814919135568967963246793041037355628136358625187316264989132993411 719-821530749276664167404164105347348417771K106356101331212250485515295-6061988412183882077651203395142455016249762755758024233918554183734543213325082458620117789262208573603966274945368255034521443161164178149284315R-357115 354-6066275633414466232421138 226385202753171477393287413593952-67186159839195850337061924350760219212534927285299243142346714905663773893365-149366203144462-334-F2 Tile 2 heatmap of counts Location 218219220221222223224225226227228229230231232233234235236237238239240241242Mutation DVISSFGGKTSYDADNRPLLVMRSN*292170121115 174216177114 1552437816782983641022922474258170529446223102120F218258153185115 -5010663165181996695290106089941342730176439015321991W263203114 97103213156250972087979981313877768314131371404543641926788Y3371591291461082969912169258114 -112 118 3601013985503161176605411 200247207P233220110 2202771869951865431617687147324848855-135323522356251150126M2261911937812516985106128395847780141377991913474170174521-169140106I279315-877637868113 107486968366108292125889645418932579965421083199L235230116 1072283689282802747493116 161380789988512--533585371111103V361-243110 1272231713317432681721852526018701010370226165-585184136111A338306122182202224151218595081019580-50384196045916819887837719017379G450265131116 108231--594031095948719269393189238213724187237528498112 C284218175131121257801845741312216712616643810689724811712846153791003142121S251255152--2856422592598-67872204611212892690242236724455256-206T28823423113013517376107110 -1551021433674401041751631146230710464220108105N377237246118 72171916323245410793164202783-889481156227636370259102-Q2241951219716821610181113 3751008883111475636957452130166626424186126112 D-36314012510920913734179253113 108-207-1149 1001441157203876463216122239E42727218710224621423323631635062521541495101127 96349614021065945022312666H399258172108101190118 12787400132238891654289571271516149252485511 65496111K3171882029412319112487-436111641061573971129 946498174182660559165112 593R26029216270110 229175116 1554459982120210450996-693192416728885-12587050100250500!"#$%&' ! 153 *****Figure B 5: F3 tile 1 heatmap of counts. *********F3 Tile 1 heatmap of counts Location 246247248249250251252253254255256257258259260261262263264265266267268269270271272273274275276277278279280281282283284285286287288289290291292293294295296297298299300301302303304305Mutation SGYDVDGTDQTSLGQFSGRVQQTYKHSVPRFFVPEHGTMFTLALVRFPPTATKEIQYLNA*151223117 9984681048156276181381035622625722855987912527666671001021052159172543417938108118 78939588466113410232832136184649200114 2402568329412014617020F1551671771091376677119 59167168810959114 -330661441534819610710932561541248977--16052891215713098-6510310516933535-36506515113121314514417242622418730W10721414198785968119 6316230708140144204164447180432301474349441012685760802018041811021111081056851281898834272904837541771271761646030769112 14026Y163215-17412295751698119325816843154264281871301256518574-4512017816075566330143339020378100741016152116 1392993410336547415712025914378227-17016619P1612588110597498113878174531887378174229264501016928122711576766115 128214-686822137-117 1211551308584789123717830951114 --7521712820817378341110 17312227M12521597114 131528710887152109901086915625925865137944721510558767410430778113 522518534918295143-113 50661539824864130395250227134200184111299847816620I13013410476115 79821578320824117 93471472722646112717466146140306910076210847170401724580919215314213315274112 17831430983557101161161216150-1846221313015L140256138869280101917419728171-593422802033622690112 23990493911170273196108119 117 13712969175909814712457-187-30979185344133792361061722178532163-18625V114 257102122-747149112 1261994185100135120205203105148-471629749427964-52735368-46125104161132184103716758385-30116 45566247886110 213119 2817914213274A9324412618215619809124182971756614060131107234182659622478221164824661118 3677139372023744110 94181212986914463-9036662994561111-1741902257029277154172-G86-10315495197-75185209494874-114 208197-88101652636251361301573064257893313222182132-134113 636549237752645097295674203104161232403089619822040C1432531419213412072125892043914789104131238228118 2056450235106102518514428438112 60451525585187198162938810556247110 299158801659521961242192097229718116621233S-2892129710087741868613434-157160191310-108807468191175584670-21341492113 110 18175101176238214108161116 342688128975126251125962571982512136131314421224896T179218991301239691-83166-130115 7314626521865599454163-5853114 151236137638024178756912775-13493-39180131317229289129-270-199145139253109163204109N10925211119312314199373191203359714050117 25426749948554181113 93163595115 197526370361985212718194147108110 12253130143306389367669922016123219780292141175-29Q149241115 110 1098410912162-27619452-1812573413278--7761445140592359059501916545931221211321219712055223623434413991907022512020723055-90116 14816D118 472111-173-117 120-1653310583376992502661391421768216710380541311202707947482528245182146383121127897154250733633311129848022199133168902657417821977E140247150218689416892109218299880621301852334984917419010734112 115 1662976439823015144-1051259197110 6051269702914410329558429194267-612418919016638H174264192739491911238216937858952220234289512658714222310318580-119 1878821881281488789-111119 139114 517512415331415912741119 741921352451818538617520517125K1281709197114 838612058219630145103521802702685178116 1252179449-86991905168442818045159121911311079655461221202562677163768216113 -20491250112 16825216R13330414012612010417917363194231609010918920223864-9515029714964110 14912622774-66281847195212133167117 113 8860225136301-1364466611981091911947550612720517159F3 Tile 1 heatmap of counts Location 306307308309310311 312313314315316317318319320321322323324325326327328329330331332333334335336Mutation KGALTYTDIAGDPVLYGNLPPREISMKDVFR*13110410110269115 113 1291022001929047401429887111336177797421971207100103691027225F105781291687710195671561951738550792296866113 40114276116 111772406551102164-41W84118 6912369107138104101141154855735171509313927013589113 854321373536613110956Y77779810185-1241691272092082145831124-75130337179939410458312775329112313328P647812113684981527414519116388-3516750112 87297--136995436994104135765983M116 69691607796114 104123246201994531187488813035116075125129315235-10688114 5239I114 9110615213565309112 -259210116 5581158415216438412568115 87-2272846184172114 34L9068125-122811251079421315410113131-7099107-70123319010687270116 72110 156279117 V721441581876458110 13514928122120649-174431711543971558081175112 20518364141-23553A73123-15326571220154147-19597826017734120100322191234901735526765761201968363G113 -161886080146172135272-139693213057-12131414357110 211 63226105562364977980C661601471369377146841372392986364191841591621562651555056110491338896214092366230S1049419618015574181115 1722573701041295624761195186371995558254108425-8486113 127219140T8267156120-51-105167271251931914816885113 14434922917110412288351142112 108102110 27N17949112 139769320820815520421822252201649090-295134779590169252972512721458928Q7080841497162115 157111224166113 566155481011032612982601051236024070921221394461D91164151119 4991154-121311 288-581011645743515229820078103160942519734-4017923E1981241051208070183169147247242161532812836981403441614985-622178511126318510427H112 79142105110 111180130148238165117 123311451228496290206878319181296598215713681220K-7392122778812218111120323187442914146124174311 16677117 24256176165-75118 6929R1117499114 11163148113 144212219105735215041139111278274421-117 53278188153139112 105-050100250500!"#$%&' ! 154 ****Figure B 6: F3 tile 2 heatmap of counts. **********F3 Tile 2 heatmap of counts Location 337338339340341342343344345346347348349350351352353354355356357358359360361362363364365366367368369370371372373374375376377378379380381382383384385386387388389390391392393394395396Mutation SGDSSKKFKIAEGQWYRYAPSYVSPAYHLLEGFPFIQEPPSGDLQERVLIRHHDYDQCFQ*423262125143109725665905220532116 19370639644122156123119 89162392073713425139491526950711231511482598873365062426425919416323116114455171252107113 9554152F97342983155110 24-1114150114 31646856489455118 15010614312212952253601506212327-46-1023688118 246677646138692021731541932179273691722429980116 -22W613544173150133277640109571512546-29577045119 1458413964112 81205621432610439119 89814245741692403810741113 461721711731731892031316415920882961095034Y116 3491611879432321617368151306595-64-54104116 -12412615688-121155239737195551355747110 114 2836793796646205259190118 267119 193118 300-142681259433P1212636244251136317640995415032908821796593-21956112 133-79198681849810838142-956555105--906745774417423020120015918312578206273112 114 663161M5718401299913193541021695713627788754114 69639513357113 6815574182581401495649163668852111163259548857122502012271811382041641318518328710078817017I36315042119 112 246512-8312642437535607047112 1221031688515869173631767497451476865-448712520368105374462172195261172-99110 59187228118 88846120L692351328173182251402412256117 1713772626906084183211 611236316873195143--9434255118 182869099285270678332-105162312262-19521918596181250149113 8219566V46847413910210435124251892143038571733558641364112 14157-6712813019179117 261741111057570119 491611872334569759540241156-16426815710662193202159116 64694A806988197122110 405634155-181525398197151-10215482190102151-1888510527115 44123111546633114 22919662103561003616312624518414614210455137287129132684425G45-7210114813344472316377210-59180158142109103114 7417476162107170889233176-16060104928017314917740-7584562101851662052401921226120920228195912911C789949103159122257815187113 1271526616782112 94739714515112295112 55243601042013388156609381761021642648119245574816533617115215643412788218271135133-9219S-10942--13648102181111701229358115 3110071100196-87129-194128208688726127106226105137656387228316-1698117971202253203166187219147622562601591111631399T6432409413714589431041741771091939109305961149185152118 13087250149218657637113 56132119 611484514227232958726356511642792171603281301827528025816985715512N443278587516961361021887916343498055778231135126531235014649216999730101501204881664210918425854777774601732032251582431372031772762742001211015418Q321350176131241885049917915238-95377585801461278416283136372257213235112 451201208861-117 28427660856199-15922221515216616013213022826591-9750-D78169-110 13991469319188185132649175523975649014710286212891541471829912926137118 112 6268112 4315117427478216-837325917922112618916514790-250-112 804410E4349115 107117 2136447135144108-248410443110 6189116 114 10317382167691809910426-6314664757359-1672576785644898-2282371412672221558216923721981848138H5616798913112820643013585117 281268418218796922071352611398821861260-19664933513378709213910913833461865677132203396209147206284--2373331461319037104K5034315790--35-1237521426114 944793675613013682117 83176691816112630170511495583117 9019013323588923359114 345243194101261177119 85123279123124675235R4949241371611729266106138751022814520825-5049123125701726714780222211 163331066111174775322984222279591573578318176-134184178-21910219725317016911166168F3 Tile 2 heatmap of counts Location 397398399400401402403404405406407408409410411 412413414415416417418419420421422423424425426427Mutation SVQLLQWNSQVKFNVTVYRNLPTTRDSIMTS*24431007827456225141571046186719851117 28732399133639824920910012459928124681184F5894221426924409938599387-5938632961939114077105202176142127260141167162101W205445512429-134678063421725689116 18623214916492118 1661871239322796251104151Y83481966115460179343666538215747119 262-12917171113 22517412512726610116514682P903957772748521324510274631038063170231162151159181-19733416691301109217181250M14865010769347612561100618585328396318266143211 141122222167152121295403-186177I251772468153458177905913390721283525145622274163961063252387111325-37924173L184082--6710412042109117 641453410189258194247212-466179163312105519118 275138214V20-42911230651267372-1089365-89-262130171115 16422214415816023112534293162A31141248938384613853741281031059413819730122715414494213401376124116 38171151430253G286816512320421447631658812417894193182146174180109133191156161162306146198167123C523828992426152126884756741399489114 25324467120464148166163259105198119 262211 121S-5836125853354196-5989711571666321823128820423279867411 804238101-1562481054-T87563960195957114 716285629312535-29826113616891223--165108345184265-127N28533293213775-715480151119 -57363254299110 -121157235208149192318170219189102Q1937-8419-7110860-4096113 90351381936856172127256169162133112 268115 230135143D3067206112296324589338490571957310219823813533813186175197102-235101216169126E334437110 403056138656294183861491301012402541511889791216226125161342862438599H22517572269363128661736088751654510024179625205116 146213189197124318110 26115583K226844631568552343915845-52135351472552406534610996180218117 95288106274118 110 R37581458422178176176105145781369012769149256168-178110 183208175-89311 126307150161050100250500!"#$%&' ! 155 *******Figure B 7: G1 tile 1 heatmap of counts. **********G1 Tile 1 heatmap of counts Location 234567891011121314151617181920212223242526272829303132333435363738394041424344454647484950515253Mutation FQTFISRHNSNFFSDKLVLTSVTPASSAPVLQTPKATSSTLYFDSLTVNAGN*298947345352823239650711408267343580110 29861021495743195754121329740313741642431726716520913215745212272334115 1008372024322635F-306-1041083021286436--8769103919512934110 84128464310721354108523902142674393842021781631015935169220-1421251771269721394249W357881444828581320924299195316137109108115 3583102126456367635442114 643451322613842751622451201896072151209379208268163293430382820Y7340111797262201295760591321721331441643100953413694118 46138075840296713531482694403982262081491077473144-4952429870132103362575P1810232693110986432520159821091367220341128286131119 98159-355663515147-351198430491-199207243235141112 143213290117 100871352911682129M44851675792726254743909013255806539599117 6184115 1264712816055778952339205305511 36022220425616246204118 2313371332121784641923313999I733054163-31202387461031282136473125531214510560182223493320630348827844019630853734820721420610351521422514381692495813773964522102L101165161786837881202413239327209636130-112 -5492127132599361658587147103369-49349342418919020230012592-207415107106-129017212233V92479107101312616306547116 1417114838272-1345773-13447747662050227956-15530730030018433813016162114 15118142140320720314-1434349724A236963140386358172899658718384116 291292109713477182244539-615381-53407233319493371183-4063039220615018844033316018344810023-6526G3344231035040951524758912817963181257319713538761211094093955574151606741813536745232119733612612593509125539438329396819715233-27C3038131993855199183995108155168897613134119 10871109106105514435667366151683291343254543172292651501736556124358558199223110 829174362239S80435613466-1263653-224162214-571953100185162-117 206553457--15684411 229345587397280353366--116 177280439160-13766049247453544169T3225-778061382540152182488118 87541037106156-115 95-697448767457692130299158377-398275242-171153-15116027117410674-33721272265N4352146881185255-60-931417912848241001516694112 15753432567641990593391542944703563501722131118065102223385205187611531-3727-Q41-76831237670389274116 13254113 40274112 126385215113149231860954813877312199-550331271183175160537315017532417919913642829223444D4242241095238412691112 85149207111-1447237116 391011841434595235874957596439158304452368269437186975956137278428-181576163139828143128E3157191224433331129134659217062214116 26166131478010512665935565534314950354167319534311 33730225910187701742084473371547286433442944H5010026865541280-499255961469813410172112 2206312291142558380725383118 15434518140953944128627419112255861023003972471526363352382375K13331575331422124851114 851405667-3182905374831635453046833369366344160361580296-169347102567914919929712315548303291175 2844645R3418516943537-993575101941445284522991042245472111116 531412646403137113 3561455425364042603361831365590126216403156201841992326698634050100250500!"#$%&' ! 156 *****Figure B 8: G1 tile 2 heatmap of counts. ******************G1 Tile 2 heatmap of counts Location 555657585960616263646566676869707172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110 111112 113 114 Mutation GFLHCIQMDTSVNAANQVVSVGADIAFDADPKFFACLVRFESSSVPTTLPTAYDVYPLNG*462566426428223934035840222225467162105112 150178257112 238121311 203297117 89195648623836016227622034235313288145384249181576177326406483142309228272304359484229402150244128107F44-139186221294153344423134264671477714813798216100288154313166223220106-7566190447136--59360297333118 -24119251212238140544412741719428923229048622229887239121111W96301100232292261379367479162305321438612593122145972301113381222701601401808294242411 17028120746236242198165258303186526147305451487113 3491632432682533492103429619578344Y252929729429919221242852117129657206781551661002409728612036821229715796187748024441014327720535272227311 129429203223543141334418450177384226312245-453191-117 193206193P59204225295215199451432445263356521259417018918725713629510335523030812115019551117 220-19737417691278288255222270252239657215349-573168423-307287268539322372-24796123M66299117 255172289363-4501502785814865166141112 2269825613030216127114310123060892764612373381907125929333814427922616857513433550151519340623427425429147322133212125498185I61274121190162-24148745422626916921310016619512133597262199376148255-95231449421338813828823447203228366154366208212493124409454483215382191413237276461292291142170146189L55381-34116718544349944719725959115 52150138213232119 230117 318167299260123300408023851813744824971260-294234416267163586280390679455195-370254293287408231384217-82141V127261145276244307279461475143266-182129163107133--214-322278365191235203649273593812093712191166 272275-173326344180629162-516505154345205248433262542-32311124285436A112 273152237293250340483468273318124157--10299197192276200485-329143-17662-276472222441303-257305361135278275182613160359492673284363228388-26343329233816923696384G-27410223127321625549144618629912316814915515184228136301146-212350127142225150147295526224414224116 224280257173258301143540154288513497158358181220322231472277345132223107-C64223101221-26832351448921730278173119 115 100101214832901333362052761481741907312421454017937024057-35933440332823424155019632448951619642620633428132048025040413622699256S12130593229347244278488464288-90243159178194114 239118 -16243327728516218924474201217591175404323132269338309216318239---288627538331414217328325285466252334197302195288T75289117 234269238220474418-3518615821945218714829711135815536628628223320723746285307474190408190169244289311 189346249240573155322446--407253-399296507284365144219130110 N872099523420625525338851020729480-134168-1352611283411723461663201581052377612136948630336019991298279322159294240199532135328583567221403238298305350548222316135230-138Q76269151374235179-4334241822556517572160187-260117 2951433562272941361401777111128043418744916636194277280157303283178569159329554564204400258337329291482228395161247150209D124243120294159266214357-178243139296122203195101283136299172519369-151174179-118 -48114729521486186290339140386366215576171384564404146350254211 298363-255287129204297464E8321911127125926328632656620926682147106147153131215118 27216537018330715313916510714335339726834526055165374328143259-184518152384466460164387186204348302530241315120161128320H52282199-17418539442660321930576175118 12517818935188330122382230281200113 21874116 25558616629821645224299302290361243207554137297423425167364213384268388560239416686182141181K56260119 255179234245340460188285892011311312422272511262651382731482781351042248179290389-28324464200256300148318280197427130315453516220345197306321270339187333120195208135R752601643443712184764124962232979213696123141250259992711113912443121951721717314425456621345128252315297363-32928519052618932854759623344524432530226556627733617524586275G1 Tile 2 heatmap of counts Location 115 116 117 118 119 120121122123124125126127128129130131132133Mutation RHDGGYYTVKDCVTIDVLP*191239696200139334349372223984372187579315193266231F5127105651221472712903781627032125695125190164384223W21118 958024710330534537717470303259116 109220163263209Y1022612168154--344310183135436244120121259176286232P50123107552091312833683732178634424315249216168372-M22997268214127270401314235105325364119 110 194193235222I14102927216412026552646617264353239148-279275327223L381711078325612729731335028180365225123103205126-276V2386121333266105304378-203145306-119 140517-304193A27981088129014129552351617913032137725992344317205274G47121111--1464133896511841503272718546582235232239C2651217259237205369416439234113 -233113 97306196279218S113 106115 114 27114425854540120387368230209123323247277322T5215410363168111354-402234107410271-148332239292310N32182118 70183131334446417247165392231185135376232287264Q34149838918214835536935022910434429314572301182249262D14136-122287154334425426207-34425510885-301308234E8114 1449528313432835839725713236132312178335247293209H144-113 68192215352363311 2219936421712680274189406299K1312611187175107253418382-99320271135105277223265260R-25110655335183299449383251117 421181118 63339171461238050100250500!"#$%&' ! 157 ********Figure B 9: G2 heatmap of counts. ***********************G2 heatmap of counts Location 136137138139140141142143144145146147148149150151152153154155156157158159160161162163164165166167168169170171172173174175Mutation PGNNVYVGFMVWSNFTATKCRGLVSLNQVIKEIICLQPLK*8807257853306861018769660607348822415041638394225996456517063908443601176 4864578797569808291454576458232172174240164252522F6235487822435851000767771-372147182477156-378444713421607311 627311 1175 4643048715739878421018448683525154183112 72293386W7247086122767889346971100 59250886-3621358903915797005197132816453278416014458509088456911128 1060383215524135278135203467Y733604895380720-7936685544251092154931759844395636795516653856653221140 5373091121 66110507571218408536426202140120152261367P-7576892777768828229506105139229356818496449654385952956032963837810787157029361093107375112631015442256118 326361-292520M878700894250765946743866544-107259400184100139052582362769235270336510906555851110 890109410221175 1039834566164213291158255527I716648825454722970830753550660158181405142107254459459455456134465827812174463881147 6631163 -1406468--14624812266323485L9107717233586829627528396426681012434661551009529538751467608352789-1143 704-8601192 9608341252985488222136-511 218-515V776664743243-803-1196 622700-311 360149870425737709570660389795411 -568586963735-8411177 1605511 345167195273122315526A9887637403169569318381292658611 1422814281751054547-8465376763316662701152 7404949837851136 911 12651648482266201160267177201538G614-655249762915790-673518128265331211 880473674690629757386-3201132 5503699018811182 82212651245472184223241194116 255464C8391133 7923467991032797944673550100337451217942517573794546-513649396123853842193283510679031258841495281-251221160251474S811 100188435279291479387875848693243-2609266139167765437604928653191324-3621133 7211140 9311540893513348190275291197270542T101589288214877491093794854699469742085302481051-581-52163743374531412345322971139 8139607551333608474249146197144166256472N935809--7471160 84181668338496133480-10065485517835805663837752951170 522334-93110778451661603501285139252136106249474Q1125 8088083208188727398475345678326548116789251253278554961834772232210866936841021-106183112921229381258138301-233309494D6919889073687681105 9581141 66547916923036919695342877576762061734471927212365593281076796123386513931195 568411 174160186104286460E71370176732694010098361010613427108196436183944421647781598721431911 2861191 514330102574110728781544-479334181201180174224480H90571877639379599586681558647913016148117610604005307105355694787862521198 4455201154 102310657841097477621316111292419179368413K78378691333875094174979963040596215400218866440577670-5874168723661093511 2831140 752917832-572487267115 213176137255-R848907842380809883807859668459110 216502223969540655783501813-838277107664154694710331125 9301429839508260213504353200388498050100250500!"#$%&' ! 158 Table B 1: Mutant library preparation summary. Summary table of the transformants required for sufficient library coverage, transformants obtained during comprehensive mutant library preparation by nicking scanning mutagenesis, and the fold excess of the number of transformants required for coverage. *******************************Gene mutated F1 F2 F3 G1 G2 Number of Residues 95 144 182 132 40 Transformants obtained following NSM 620,000 890,000 370,000 620,000 640,000 Required transformants for 99.9% coverage of possible library 13781 20889 26401 19148 5803 Fold excess over amount required for coverage 45 43 14 32 110 ! 159 Table B 2: Primers for incorporating BbvCI nicking sites into the pCR2.1 -topo shuttle vector. Blue text is the overlap region with the shuttle vector, red is the KpnI site, green is the BbvCI site. !!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!*Nick_incorporation_fwd GCTCTACG GGTACC GCTGAGG GAGCTCGGATCCACTAGTAACG Nick_incorporation_rvs GCTCTAACG GGTACC AAGCTTGGCGTAATCATGGTC ! 160 Table B 3: Inner and outer primers for PCR reactions for Illumina sequencing. Red indicates overhang regions for attaching Illumina adapter primers (inner PCR primers) or overhangs for attaching to inner PCR product (outer PCR primers), black is the overlap region in the gene or the barcode, blue is the Illumina adapter. ** Inner PCR Primers Sequence (5' to 3') Fragment F1 Fwd gttcagagttctacagtccgacga cccttacttgaggataaatt Fragment F2 Tile 1 Fwd gttcagagttctacagtccgacgatc gactcactatagggcgaa Fragment F2 Tile 2 Fwd gttcagagttctacagtccgacgatc agcttaatcaagatgatgct Fragment F3 Tile 1 Fwd gttcagagttctacagtccgacgatc cgtctctctgggca Fragment F3 Tile 2 Fwd gttcagagttctacagtccgacgatc gaaggatgttttccgt Fragment G1 Tile 1 Fwd gttcagagttctacagtccgacgatc gaattgggccctctag Fragment G1 Tile 2 Fwd gttcagagttctacagtccgacgatc ggttaatgctggtaatgg Fragment G2 Fwd gttcagagttctacagtccgacgatc ggccctctagatgca Fragment F1 Rvs ccttggcacccgagaattcca ttcgtctcacagtcgg Fragment F2 Tile 1 Rvs ccttggcacccgagaattcca caacggaaaccataacg Fragment F2 Tile 2 Rvs ccttggcacccgagaattcca ttcgtctccccagag Fragment F3 Tile 1 Rvs ccttggcacccgagaattcca tcttagacgaatcaccaga Fragment F3 Tile 2 Rvs ccttggcacccgagaattcca tcacactcaatcttttatca Fragment G1 Tile 1 Rvs ccttggcacccgagaattcca tgcaatgaagaaaacca Fragment G1 Tile 2 Rvs ccttggcacccgagaattcca cttcgtctccgtacg Fragment G2 Rvs ccttggcacccgagaattcca ttgaccgcctcca Illumina outer primer adapter aatgatacggcgaccaccgagatctacac gttcagagttctacagtccga Illumina outer PCR adapters and barcodes RPI31 (Fragment F1) caagcagaagacggcatacgagat ATCGTG gtgactggagttccttggcacccg agaattcca RPI15 (Fragment F2 Tile 1) caagcagaagacggcatacgagat TGACAT gtgactggagttccttggcacccg agaattcca RPI16 (Fragment F2 Tile 2) caagcagaagacggcatacgagat GGACGG gtgactggagttccttggcaccc gagaattcca RPI17 (Fragment F3 Tile 1) caagcagaagacggcatacgagat CTCTAC gtgactggagttccttggcacccg agaattcca RPI18 (Fragment F3 Tile 2) caagcagaagacggcatacgagat GCGGAC gtgactggagttccttggcaccc gagaattcca RPI19 (Fragment G1 Tile 1) caagcagaagacggcatacgagat TTTCAC gtgactggagttccttggcacccg agaattcca RPI20 (Fragment G1 Tile 2) caagcagaagacggcatacgagat GGCCAC gtgactggagttccttggcaccc gagaattcca RPI21 (Fragment G2) caagcagaagacggcatacgagat CGAAAC gtgactggagttccttggcaccc gagaattcca ! 161 APPENDIX C Purification of the TROP2 extracellular domain from a stable insect cell line using ammonium sulfate precipitation ! 162 Abstract The tumor associated calcium signal transducer 2 (TROP2) is an oncogenic transmembrane protein that is overexpressed in aggressive and late stage cancers. Here we present a facile method for purifying approximately 3 mg/L quantities of the extracellular domain of TROP2 from a stable Drosophila SchneiderÕs (S2) cell line. The secreted oncogene is purified by ammonium sulfate fractionation, immobilized metal affinity chromatography, and size exclusion chromatography. The folding fidelity of the highly purifie d product was confirmed with a binding assay that tests for a unique conformational epitope in TROP2. ! 163 Introduction The tumor associated calcium signal transducer 2 (TROP2) is a 36 kDa type -I transmembrane glycoprotein associated with late stage and aggressively metastatic tumors 1-3. TROP2 is basally expressed in human trophoblasts when the fetal tissue joins into the maternal circulation 4. Ectopic expression of TROP2 in cancer cells results in increased proliferation and tumorigenicity 5-8, while gene silencing of TROP2 decreases these same characteristics 5,7,9 . Following an activation event that is currently unknown, TROP2 undergoes regulated intramembrane proteolysis 10 at two distinct sites to release the extracellular domain (TROP2Ex, resi dues 27 -274) and the intracellular domain (TROP2Ic, residues 298 -323). TROP2Ic migrates to the nucleus where it increases the expression of genes associated with cell proliferation 10-11. The liberated extracellular domain interacts with signaling proteins, though with which ones and how is incompletely understood 8,12 -13. Previous biophysical analysis has found that TROP2Ex can assume a dimeric conformation similar to its paralog epithelial cell adhesion molecule (EpCAM) 14-16. Interestingly, the glycosylat ion state of the protein seems to dictate whether the monomeric (favored when fully glycosylated) or dimeric (favored when unglycosylated) form predominates at equilibrium 15. Previous work studying the migration of metastatic cancer cells found that the oligomeric state of TROP2 might govern its function 9. Whereas binding of TROP2 with the bivalent m7E6 monoclonal antibody (mAb) 17 resulted in significant inhibition of migration of MDA-MB-231 cancer cells while not impacting toxicity or cell proliferation, b inding with the monovalent m7E6 Fab did not significantly alter migration. These results suggest that the dimer to monomer transition of TROP2 may play a role in its activation and mechanism of action. ! 164 Here we provide a novel and facile approach for the purification of properly folded TROP2Ex from insect cells. We used a stably transformed Drosophila SchniederÕs (S2) cell line to secrete the protein. The secreted protein is removed from the culture media with a classical but underutilized ammonium sulfat e fractionation method. Next, the protein is further purified with immobilized metal affinity chromatography and size exclusion chromatography. We obtain yields above 3 mg/L of induction culture with excellent purity. The correct folding of the protein has been verified with flow cytometry binding assays using a single chain variable fragment (scFv) of the m7E6 mAb displayed on the yeast surface, which recognizes a conformational epitope in TROP2Ex. The purified TROP2Ex can be used in a variety of in vivo and in vitro experiments to help understand how TROP2 is activated. Additionally, the yeast displayed m7E6 scFv system presented here can be used to map the paratope involved in the TROP2 m7E6 binding interaction. Results and discussion Expression of TRO P2Ex from a stable cell line As TROP2Ex contains six potential disulfide bonds and multiple N -linked glycosylation sites, we selected Drosophila Schneider 2 (S2) cells for our expression host ! 165 because of their potential ability to accurately maintain these post -translational modifications, their low house -keeping requirements, ease of transfectio n, the advantages of protein secretion, and the ease of forming stably transfected cell lines. The TROP2 gene was truncated to residues 27-254 (TROP2Ex) for expression and secretion in S2 cells ( Figure C 1A-B). The native secretion signal peptide sequence (SP) was replaced with the secretion tag needed for expression in S2 cells (ST) ( Figure C 1B). A His 6x tag was added to the C -terminal end of the truncated TROP2Ex gene ( Figure C 1B) to facilitate purification. !"#$%&'( )*+(,'(-!( .(/(!"#$%&'( !0( !"#$%12( -$(.(/(.34*567( $89*:*6;7(/<4=+=>( &'49326>>8>39(+?326( !"#$%&'( !"#$%12( A. B. C. $3@6"8>69( ?96+43*A6;(>3;;69( %B(C=89+( BD(C=89+( E%(C=89+( F,(C=89+( G%H(C=89+( IJ(KL3(BH(KL3(H(J(GH(GJ(%H(H(%H(BH(,H(DH(GHH(G%H(M3A;(*A46A+*4<(3;N8+46;( :=9(O32K@9=8A;( !*P6(QC=89+R( Figure C 1: TROP2Ex and its expression. A. Homology model of the dimeric TROP2Ex domains generated using the paralog EPCAM 14,16 (pdb: 4MZV). This model was previously published 9. B. Diagram of the native and purified form of TROP2, with the dashed line representing the truncation point in the secretory pathway, SP = signal peptide, ST = secretion tag, TM = transmembrane domain, TROP2Ic = TROP2 intracellular domain. C. Western blot testing for TROP2Ex in equivalent volumes of the stable S2 cell supernatant following initiation of induction and band intensity adjus ted for background is plotted against time; bands were quantified with ImageJ by standard protocols. ! 166 A stable S2 cell line that secretes TROP2E x was successfully generated as described in the Materials and Methods (Figure C 1C). The secreted TROP2Ex runs larger than the predicted molecular weight - ~28.8 kDa Ð because N -linked glycosylations can occur at up four distinct sites after translation 15. The robustness of the cell line was verified through multiple successful TROP2Ex purifications from the same lineage over the course of months (data not shown). After generating the stable S2 cell line we next increased the culture volumes possible by tr ansitioning from static to shaking cultures. The addition of a non -ionic surfactant the cells maintained healthy morphologies in the shaking cultures as assessed by confocal microscopy. The optimal induction time for secreted TROP2Ex titers was performed t he shaking cultures, with a 96 -hour induction period striking the best balance between titers and volumetric productivity ( Figure C 1C). The shaking cultures were successfully scaled to 300 mL and induced for 96 hours using copper. Purification of TROP2Ex TROP2Ex was purified using ammonium sulfate fractionation, IMACs, and chromatography. Fractionation with ammonium sulfate at 40% saturation, followed by fractionation at 65% saturation efficiently removed some of the contaminating proteins and contaminant copper and allowed for resuspension of the secreted TROP2Ex in IMAC buffer (Figure C 2A). Batch IMAC resulted in nearly pure TROP2Ex ( Figure C 2A) and size exclusion chromatography (SEC) using a Superdex 200 column equilibrated wi th 1x PBS at 4¡C removed the remaining impurities ( Figure C 2B-C). TROP2Ex eluted from SEC as a peak with a shoulder between 78 -90 mL elution volume ( Figure C 2B-C), suggesting that both monomeric and dimeric TROP2Ex exist under elution conditions. ! 167 !Figur e C 2: Purification of TROP2Ex from insect cell cultures. A. Images of an SDS -PAGE gel and a Western blot containing identical samples resulting from the purification of TROP2Ex. B. FPLC -SEC chromatogram of the IMACs eluent, column was equilibrated with 1x PBS at 4¡C , main peak and respective shoulder at ~78 Ð 90 mL are TROP2Ex. C. SDS-PAGE image of the shoulder (80 mL elution volume) and main peak (85 mL elution volume). Interestingly, when the purified TROP2Ex main peak is run again with the same SEC cond itions as before both the main peak and shoulder are resolved (data not shown). All subsequent analysis corresponds only to TROP2Ex from the main peak ( Figure C 2C). Under conditions used in these experiments TROP2Ex (unglycosylated mass = 28.8 kDa) elutes at ~85 mL ( Figure C 2B), while BSA (66.5 kDa) elutes at ~73 mL, and GFP (26.7 kDa) elutes at ~88 mL off of the Superdex 200 column in identical conditions. This elution pattern indicates that our purified TROP2Ex is predominantly monomeric. This finding i s consistent with previous results finding that only a small fraction of the glycosylated TROP2Ex is in the dimeric form when at equilibrium 15. In total, we obtain yields of 3.6 mg/L of purified monomeric TROP2Ex from the stable cell line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alidation of TROP2Ex folding To verify that TROP2Ex was in a properly folded conformation, we assessed its binding to a yeast displayed scFv derived from the high affinity anti -TROP2 mAb m7E6 17 (Figure C 3A). m7E6 recognizes a conformational epitope Figure C 3: Verification of the proper assembly of the m7E6 scFv yeast display construct and of binding to TROP2Ex. A. A diagram of the yeast display system, the binding interactions detected, and the respective conjugate fluorophores: SA -PE = streptavidin R -phycoeryt hrin conjugate. B. A DNA gel containing restriction enzyme analysis products for verifying the assembly of the m7E6 scFv pETconNK construct via homologous recombination. Following digestion the DNA was run on a 1% agarose gel prestained with ethidium bromi de at 120 V for 50 minutes, the 1 kb DNA ladder is from GoldBio. Following electrophoresis the gel was imaged using a Safe Imager TM Blue Light Transilluminator. C. Clonal titration of yeast displayed m7E6 scFv with TROP2Ex, n = 3, error bars = 1 S.D. loca ted in the membrane distal region of TROP2Ex 9. Overhang PCR and homologous recombination in yeast were used to assemble to the anti -TROP2 m7E6 scFv yeast display construct as described in the Materials and Methods section ( Figure C 3B). Clonal titrations resulted in a binding curve that spanned a >25 -fold change in signal intensity and provide a preliminary dissociation constant (K D) of 180 ± 54 nM ( Figure C 3C). There are multiple possible reasons the titrations didnÕt reach saturation: we didnÕt expand th e titrations to high enough concentrations, the yeast displayed form of m7E6 might not be stable, or perhaps the !"#$%&'& !"#$%&(& )$*+,%-.%*& *+,%-.%*& )$*+,%-.%*& *+,%-.%*& '&/0&123&"4**%5& 678&9888&!"## $"#:;<'88& =>?@(:A& B3C@:& DE&DF&GCHIG& %J+.#J%& KL=!& 4$.+CGCHIG& H30& H6:9&-GKM& %"## 010020030040001000200030004000500060007000Conc TROP2Ex (nM)PE fluorescenceN%4$&O")#5%-G%$G%& !#$G%$.54.+#$&=>?@(:A&P$NQ& 8&'88&(88&R88&S88&8&6888&9888&7888&S888&R888&(888&'888&! 169 binding reaction conditions are not fully optimized for this pairing. The obtained K D is ~4 times larger than the ~44 nM K D reported for the bi nding of this pairing where TROP2Ex is displayed on the surface of yeast and recombinant m7E6 fragments of antibodies (Fabs) are applied 9. The large difference in the K D could be the result of the titrations failing to reach saturation, different glycosyla tion patterns on either protein, differences introduced into the CDRs or framework of the antibody fragments resulting from the truncation of the framework region, or it is possible that the orientation of the V H and V L domains of the scFv arenÕt optimal. This pairing binds with a dissociation constant in the hundreds of nanomolar, indicating that the purified TROP2Ex contains the same conformational epitope as the native TROP2Ex and is likely correctly folded. Conclusion Here we presented a facile method for purifying the extracellular domain of TROP2 from Drosophila SchneiderÕs (S2) insect cells. The cell lines generated are stable and can be seeded for TROP2Ex production once recovered from storage. Removing the need for retransformation allows for an easy scale up of the induction cultures. Additionally, we have presented a method for purifying TROP2Ex using ammonium sulfate fractionation, IMACs, and chromatography. This method allows for the purification of >3 mg/L of p roperly folded TROP2Ex from an induction culture in a single day. Materials and m ethods: Materials Antibiotics were purchased from GoldBio and ThermoFisher. All enzymes were from New England Biolabs (NEB), and all other chemicals were purchased from Si gma -Aldrich. The ! 170 TROP2Ex gene block, and all primers were purchased from Integrated DNA technologies (IDT); the m7E6 full IgG gene which was purchased from GenScript. Flow cytometry probes were purchased from Miltenyi Biotec (anti -c-myc FITC) and ThermoFis her (streptavidin R -phycoerythrin conjugate). Other materials used have their respective manufacturers listed in the text. Plasmid Construction The gene for TROP2Ex (Kozac -Bip -TROP2Ex) was designed as follows following a 5Õ to 3Õ convention: a KpnI rest riction site, followed by a Kozac sequence and a Bip secretion tag (Supporting text, Note S1 ), then the TROP2Ex gene (residues 27 -274), with an AgeI restriction site. Kozac -Bip -TROP2Ex was codon optimized for expression in D. melanogaster and was ordered a s a single gene block from IDT ( Supporting text, Note S1 and S3 ). The Kozac -Bip -TROP2Ex gene was subcloned into the pMT/V5 -His B plasmid (Invitrogen) using KpnI and AgeI restriction enzymes and standard cloning methods to produce pMT -Bip -TROP2Ex containing an in -frame C -terminal 6x His tag. The final construct was transformed into E. coli XL-1 Blue and a glycerol cell stock was generated. A scratch of the freezer stock was used to inoculate 50 mL LB + 50 g/mL carbenicillin which was grown overnight at 37 ¡C. DNA was Midi -prepped (Qiagen) from 25 mL of the overnight culture, and the purified DNA was resuspended in nuclease free H 2O and frozen at -20¡C. The m7E6 scFv 17 (Supporting text, Note S2 and S4 ) comprises (5Õ to 3Õ) V H residues 1 -118, a flexible (GGGG S)3 linker, and V L residues 1 -111. Using the full m7E6 IgG gene that was previously purchased from Genscript 17 as template, overhang PCR was used to assemble the scFv, introduce the linker, and introduce homology regions from the pETconNK yeast display ! 171 pla smid18. Primers used for assembly are in Note S5 of the supporting text. The PCR products were cleaned and concentrated using a Monarch ¨ PCR & DNA Cleanup Kit (NEB) and eluted into nuclease free H 2O. The scFv gene was assembled with pETconNK vector cut wit h NdeI and XhoI using homologous recombination into S. cerevisiae EBY100 cells. For homologous recombination the V H and V L inserts where combined with the cut pETconNK plasmid at a ratio of 30 fmol : 1 fmol (respective insert : cut vector) in a final volume of 8 µL. All of the DNA mixture was transformed into EBY100 chemically competent yeast using standard protocols. Transformation mixtures were plated on agar plates containing Synthetic Complete media supplemented with 2% (w/v) dextrose ( SDCAA agar) and grown at 30¡C for 2 -3 days. Colonies were picked and grown in 1 mL of SDCAA and 1x penicillin/streptomycin (SDCAA media) at 30¡C with shaking at 250x rpm for ~16 hours. The 1 mL cultures were then used to inoculate 50 mL of SDCAA media that was grown for generation of freezer stocks essentially as described in Klesmith et al. 18. Both plasmids used in this work are available from Addgene: m7E6 scFv pETConNK (ID: 125731), and pMT -Bip -TROP2Ex (ID: 125730). Transformation of S2 Drosophila Cells and the Generation of the Stable Cell Line The S2 Drosophila cells were cultured, transformed, and a stable cell line was generated essentially as described by the manufacturer (Invitrogen). Briefly, 3 mL/well of SchneiderÕs S2 Drosophila Media (ThermoFi sher) supplemented with 10% heat -inactivated fetal bovine serum (full S2 media) was seeded with 1x10 6 cells/mL in a 6 -well plate (Fisher Scientific). Cells were incubated at 28¡C until a density of 2 -4x106 cells/mL was reached, after approximately 16 hours . After reaching density the cells were transformed with 19 µ g pMT -Bip -TROP2Ex plasmid and 1 µg pCoPuro plasmid 19 via standard calcium phosphate transfection. Calcium phosphate DNA ! 172 precipitates were prepared using the Calcium Phosphate Transfection Kit (I nvitrogen) as described by the manufacturer. The DNA precipitate solution was added to respective wells such that 5 µ g total DNA was applied. Additions were performed in a dropwise fashion with gentle swirling of the culture after each addition. After addi tion of the DNA precipitates the cultures were incubated at 28¡C overnight. The next morning the transformation cultures were spun at 440 xg for 3 minutes and the transfection media was removed via aspiration. Cells were resuspended in 3 mL of full S2 medi a and spun as before. This media was aspirated and the washing procedure was repeated once more. Cells were resuspended in 3 mL full S2 media, placed back into the original well and incubated at 28¡C for 48 hours. Puromycin toxicity was assessed as describ ed in the Drosophila S2 Cell Manual (Invitrogen). It was determined that 2 µg/mL puromycin was the optimal concentration for selection of stable S2 cells harboring the pMT -Bip -TROP2Ex and pCoPuro plasmids. Following the recovery of the transformation cultures, a single culture was brought to a final concentration of inducer - 500 µM CuSO 4 - and incubated for 48 hours at 28¡C. After 48 hours the culture was spun at 3,200 xg for 10 min to pellet the cells. The supernatant was filtered through a 0.22 µM P ES filter and stored at 4¡C. A portion of the aliquot was assessed via Western blotting with standard protocols. Briefly, the sample was denatured in LaemmliÕs buffer supplemented to 1.5% *-mercaptoethanol at 1x for 10 minutes at 98¡C. The denatured prote in was run on a 4 -20% Mini -PROTEAN ¨ TGXª precast gels (Bio -Rad) for 1 hour at 120 V, and the PageRuler Prestained Protein Ladder (10 -180 kDa, Thermo -Fisher) was used as a ladder. Once separated on the gel the proteins were transferred to a nitrocellulose m embrane using the iBlotª Gel Transfer Device with iBlotª Transfer Stacks as per the manufacturer instructions (ThermoFisher). The primary antibody was Anti -6x His tag ¨ horseradish peroxidase conjugate ! 173 antibody (abcam, ab1187), and the blot was resolved usi ng the Pierceª DAB Substrate Kit as described by the manufacturer (ThermoFisher). The generation of the stable cell line was performed in parallel to the initial testing of TROP2Ex production. Recovered cells were spun at 440 xg for 3 minutes the superna tant was aspirated, the pellet was resuspended in 3 mL of full S2 media + 2 µg/mL puromycin and placed back into the original well. Every 3 -4 days the culture was pelleted, the supernatant removed via aspiration, gently resuspended in 3 mL of fresh full S2 media with 2 µg/mL puromycin, placed back into the original well and grown at 28¡C. This practice was continued until the cells had grown a density of 6 -20x106 cells/mL. Once at density the cells were then passaged 1:2 into full S2 media + 2 µg/mL puromyc in to 6 mL final volume. At this point the culture was moved into a Corning ¨ 25 cm 2 culture flask with a vented cap (Sigma -Aldrich) and grown at 28¡C. Once the cells reached a density of 6 -20x106 cells/mL they were passaged 1:2 into 12 mL full S2 media with puromycin in a Corning ¨ 75 cm 2 culture flask with a vented cap (Sigma -Aldrich) and grown at 28¡C. When the cells were passaged into the 75 cm 2 flask a 6 well plate was also seeded with 3 mL full S2 media + 2 µg/mL puromycin with 1x106 cells/mL and TROP2E x expression was tested as previously described. The stable cell line was cultured in the 75 cm 2 flask until the cells grew to 1 -2x107 cells/mL, at which time freezer stocks were prepared. To prepare freezer stocks the cultures were spun at 440 xg for 3 minutes and the conditioned media was collected in a 50 mL falcon tube via careful pipetting. Cells were resuspended in 10 mL room temperature sterile 1x PBS (10 mM Na 2HPO4 + 2 mM KH 2PO4 + 2.7 mM KCl + 137 mM NaCl, pH 7.4) and spun as before, the resulting supernatant was removed via aspiration. Freezing buffer was prepared by combining 9 mL conditioned media with 9 mL full S2 media and 2 mL 100% DMSO. The cells were gently resuspended in the room ! 174 temperature freezing buffer at 1.1x10 7 cells/mL and 1 mL ali quots were dispensed into cryogenic storage vials. A freezer box was lined with paper towel and the samples were stored therein, this box was placed into a Styrofoam container and this was stored at -80¡C. After 24 hours at -80¡C the vials were moved into liquid nitrogen storage. The remaining culture was continually expanded via 1:2 dilutions into fresh full S2 media with puromycin as before. Once at 30 mL final volume, cells were passaged 1:10 into full S2 media with puromycin every ~6 -7 days. Production and Purification of TROP2Ex: In a sterile 250 mL Erlenmeyer flask, 50 mL dynamic full S2 media (full S2 media + 2 µg/mL puromyci n + 0.1% Plurionic ¨ F68 (Sigma -Aldrich) + 1x penicillin/streptomycin) was seeded with stable S2 cells (pMT -Bip -TROP2Ex -pCoPuro ) at 1x10 6 cells/mL, this was grown at 28¡C with shaking at 110 rpm. This culture was grown to a density of ~2 -4x106 cells/mL, once there CuSO 4 was added to a final concentration 500 µM. E xtractions were performed every 24 hours for 120 hours following in itiation of TROP2Ex expression. Collected samples were analyzed via Western blotting as described previously. For larger scale production 300 mL of dynamic full S2 media was seeded and grown as described previously in a 2 L baffled culture flask. Once a t ~2 -4x106 cells/mL (~24 -48 hours) the culture was induced CuSO 4 as before for 96 hours at 28¡C with shaking at 110 rpm. After induction the culture was spun at 3000 xg for 30 minutes at 4¡C, the supernatant was saved and the pellet discarded. The volume o f the supernatant was measured with a graduated cylinder and then filtered through a 0.22 µ m PES filter. After filtration, the supernatant was placed into a sterile 1L beaker with a stir bar, placed into an ice bath on a stir plate, and brought to 0.1 mM PMSF. With continuous gentle stirring solid ammonium sulfate was slowly added to the ! 175 supernatant until the solution was 40% of the saturation limit for ammonium sulfate at 0¡C. The mass of solid ammonium sulfate to add was calculated using a web -based calcu lator (http://www.encorbio.com/protocols/AM -SO4.htm ). After addition of ammonium sulfate the beaker was covered and stirred for 3 hours in the ice bath. After the equilibration, the 40% saturated ammonium sulfate supernatant was spun at 10,000 xg for 15 minutes at 4¡C. Following centrifugation the supernatant and precipitate were separated, the pellet was discarded, and the supernatant was moved back into the ice bath. With gentle stirring the 40% saturated supernatant had solid ammonium sulfate slowly added until the solution was 65% of the saturation limit for ammonium sulfate at 0¡C . The 65% ammonium sulfate saturated solution was covered and stirred for 1 hour in the ice bath. After the second equilibration the solution was spun at 15,000 xg for 15 minutes at 4¡C. The supernatant was discarded, and the pellets were resuspend in a total of 40 mL 1x PBS + 20 mM imidazole, and pooled together. Batch immobilized metal affinity chromatography (IMA C) was performed using a 1 mL bed volume of Talon ¨ resin (Takara Bio) pre -equilibrated with 1x PBS + 20 mM imidazole. A 3 -hour adherence reaction was performed by rocking the samples with the resin in an ice bath. The slurry was pelleted at 700 xg for 2 mi nutes and the supernatant was discarded. The slurry was then resuspended in 10 mL of 1x PBS with 20 mM imidazole with gentle pipetting, transferred to a 15 mL conical tube, and incubated for 5 minutes with rocking in the ice bath. The resin was pelleted as described above and the supernatant was discarded, this wash step was repeated four more times. To elute TROP2Ex the pelleted resin was resuspended with 1 mL of 1x PBS + 400 mM imidazole with gentle pipetting and incubated for 5 minutes with rocking in an ice bath. The slurry was spun as before and the supernatant was collected and stored in ice, this was repeated ! 176 five more times. IMACs purified TROP2Ex was concentrated from ~6 mL to ~2.5 mL with Amicon ¨ Ultra -4 10,000 NMWL centrifugal filters and filtered through a 0.22 µ m PES filter. The IMAC eluent was further purified by FPLC -SEC using a HiLoad 16/600 Superdex 200 pg size exclusion column equilibrated with 1x PBS with a mobile phase flow rate of 1 mL/min. The main peak eluted at 85 mL with a shoulder that eluted at 80 mL, and the main peak and shoulder were not pooled. The collected fractions were filter through a 0.22 µ m PES filter and stored at 4¡C. Successful purification of the protein was confirmed by Western blotting and the purity of the final product was confirmed using SDS -PAGE stained with SimplyBlue SafeStain as per the manufacturer instructions (ThermoFisher). The purified protein was quantified using both Bradford assays with standard protocols, and by the Edelhoch method with a predicted molar extinction coefficient at A 280 of 16680 M -1 cm-1. Yeast display of the m7E6 scFv and clonal titration Yeast surface display of the m7E6 scFv with EBY100 cells was optimized with the optimal displaying conditions discovered to be 30¡C for 16 -18 hours with shaking at 250 rpm in a 1 mL culture. TROP2Ex was chemically biotinylated using NHS -Ester chemistry and clonal titrations were performed exactly as described by Medina -Cucurella and Whitehead 20. Acknowledgements We would like to thank Dr. C. Chan for allowing us to use her laboratory infrastructure and Dr. A. Oak for her training and help with insect cell culturing. ! 177 Supporting text Note S1. DNA sequence of the TROP2Ex gene block inserted into the pMT vector . D. melanogaster optimized TROP2Ex gene (underlined text), Kozac sequence (blue text), Bip secretion tag (orange text), KpnI and AgeI restriction sites are bolded and italicized. GGTACC ACCATGGGA ATGAAGTTATGCATATTACTGGCCGTCGTGGCCTTTGTTGGC CTCTCGCTCGGG CATACGGCAGCCCAGGATAATTGTACTTGTCCAACGAATAAA ATGACGGTATGCTCCCCGGACGGACCAGGCGGTAGGTGTCAATGTCGGGCACTCGGAAG TGGAATGGCTGTTGACTGTTCCACACTCACGAGTAAATGTTTGCTCCTGAAGGCAAG GATGAGCGCTCCTAAAAACGCTCGAACTCTCGTCAGGCCAAGTGAGCACGCGCTGG TCGACAACGACGGCTTGTACGATCCCGATTGCGACCCAGAGGGCAGGTTTAAAGCT CGCCAGTGCAACCAAACGTCGGTCTGCT GGTGTGTAAACTCCGTGGGTGTACGACGG ACCGACAAAGGTGATTTGTCCCTGCGTTGTGACGAACTCGTCCGCACGCATCACATA CTCATTGATCTGAGGCATCGTCCCACCGCTGGAGCGTTCAATCACTCGGACTTGGAC GCCGAACTGCGCCGATTGTTCCGAGAACGATATAGGTTGCATCCCAAGTTCGTTGCT GCGGTACATTACGAGCAACCAACCATACAGATAGAATTGAGGCAAAATACCAGTCA AAAAGCGGCGGGAGATGTAGATATTGGCGATGCAGCCTATTATTTCGAAAGGGATA TTAAAGGCGAATCGTTGTTCCAAGGTCGAGGCGGACTCGACCTCCGGGTTAGGGGC GAGCCATTGCAGGTCGAAAGGACTCTGATCTATTACCTCGATGAAATACCTCCGAAG TTTAGCATGAAGAGGTTG ACCGGT Note S2. DNA sequence of the m7E6 scFv. The sequence for th e V H domain is blue, the sequence of the linker is underlined, the V L sequence is lower case. CAGGTCCAGCTGAAGGAAAGCGGCCCCGGCCTGGTGGCCCCTTCCCAGTCTCTGAG CATCACCTGCACAGTGAGCGGCTTCTCCCTGACCTCTTACGGCGTGCACTGGGTGCG GCAGCCTCCTGGCAAGGGCCTGGAGTGGCTGGGCGTGATCTGGACCGGCGGCTCCA CAGATTATAACTCTGCCCTGATGAGCCGGCTGTCCATCAACAAGGACAATTCCAAGT CTCAGGTGTTTCTGAAGATGAATAGCCTGC AGACCGACGATACAGCCATGTACTATT GTGCCCGGGACGGCGATTACGACAGATATACCATGGATTACTGGGGCCAGGGCACC AGCGTGACAGTGAGC GGTGGAGGGGGTTCAGGCGGGGGTGGAAGCGGTGGAGGGG GTAGCagtgatatcgtgctgactcagtcccctgcttcactggccgtgagcctgggccagagagctacaatctcctgcagagcctccaag tctgtgagcacctccggctacagctatatgcactggtaccagcagaagccaggccagccccctaagctgctgatctatctggcctctaacct ggaaagcggcgtgcctgctcggttctctggcagcggctccggcacagactttaccctgaatattcacccagtggaggagg aggatgccgc cacatactattgccagcactccagggagctgccctacacattcggcggcggcaccaagctggagatcaag Note S3. Secreted TROP2Ex amino acid sequence. HTAAQDNCTCPTNKMTVCSPDGPGGRCQCRALGSGMAVDCSTLTSKCLLLKARMSAP KNARTLVRPSEHALVDNDGLYDPDCDPEGRFKARQCNQTSVCWCVNSVGVRRTDKGD LSLRCDELVRT HHILIDLRHRPTAGAFNHSDLDAELRRLFRERYRLHPKFVAAVHYEQPT IQIELRQNTSQKAAGDVDIGDAAYYFERDIKGESLFQGRGGLDLRVRGEPLQVERTLIYY LDEIPPKFSMKRLTGHHHHHH ! 178 Note S4 . Amino acid sequence of the m7E6 scFv. QVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVHWVRQPPGKGLEWLGVIWTGGSTDY NSALMSRLSINKDNSK SQVFLKMNSLQTDDTAMYYCARDGDYDRYTMDYWGQGTSV TVSGGGGSGGGGSGGGGSSDIVLTQSPASLAVSLGQRATISCRASKSVSTSGYSYMHWY QQKPGQPPKLLIYLASNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHSRELPY TFGGGTKLEIK Note S5. Primers for overhang PCR for forming the m7E6 scFv and introduction of the cut sites for cloning into pETconNK. 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