DEVELOPMENT OF NOVEL FLUORESCENT PROTEIN TAGS FOR NO-WASH LIVE-CELL IMAGING WITH MINIMUM FLUORESCENT BACKGROUND By Rahele Esmatpour Salmani A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Chemistry—Doctor of Philosophy 2022 i ABSTRACT DEVELOPMENT OF NOVEL FLUORESCENT PROTEIN TAGS FOR NO-WASH LIVE-CELL IMAGING WITH MINIMUM FLUORESCENT BACKGROUND By Rahele Esmatpour Salmani Recent fluorescence microscopy technologies have revolutionized many areas of biomedical research. Nonetheless, high brightness, far-red/near infra-red emission, deep tissue penetration, and selective fluorescent imaging with the minimum background are among the most desired novel fluorescent labeling. One of our primary goals is to develop flexible fluorescent protein tags capable of being tailored ad infinitum. We successfully demonstrated the ability to fine-tune the absorption and emission spectra of protein-bound chromophores over an unprecedented wide range (~200 nm). In contrast to intrinsically fluorescent proteins that are always “ON” in our systems, fluorescent is activated upon covalent binding of ligand and the target protein leading to temporal control of fluorescence. However, the fluorescence background from unbound free chromophore and non-specific binding has always been a deep concern in fluorescent labeling. This Ph.D. research aimed to develop novel protein- based fluorescent tags emitting in the far-red/NIR region of the spectrum for no-wash background-free live-cell imaging applications. This was accomplished by coupling novel synthetic fluorogenic chromophores with hCRBPII mutants. Unbound free aldehyde ThioPhenol and CyThioPhenol are non-emissive dyes that become highly fluorescent upon imine formation with an active site lysine residue engineered deep in the hCRBPII cavity. We created a hydrogen-bonding network around the ThioPhenol hydroxyl group through rational protein engineering that facilitates its deprotonation ii upon photoexcitation. On the other hand, engineering the target protein to maintain a high iminium pKa resulted in Protonated Schiff Base (PSB) formation. The resultant complex experiences a strong intramolecular charge transfer (ICT), leading to fluorescence and a large bathochromic shift in the emission (~700 nm). The designed protein-based photoacid provides an unprecedented spatiotemporal control for no- wash bright NIR imaging. Our most recent report demonstrated that hCRBPII/chromophore complexes could be developed as a photobase where the imine is converted to an iminium upon photoexcitation. In the course of optimizing hCRBPII to promote ESPT of the hydroxyl group, we discovered that ThioPhenol is capable of acting as both a photoacid and a photobase upon a single photoirradiation. When bound as a Schiff base (SB) to protein mutants that maintain a low iminium pKa (~5), engineered to deprotonate the hydroxyl group, a dual ESPT process leads to protonation of the imino to iminium (the photobase) and deprotonation of the hydroxyl to alkoxide (the photoacid). This double ESPT feature is recapitulated in a protein- ligand micro-environment, yielding bright protein-dye complexes with unapparelled large pseudo-Stokes shifts (~250 nm). Additionally, the double ESPT ThioPhenol/hCRBPII complexes show fast binding rates (half-life of <3 min) that were successfully used to visualize whole-cell and the nucleus as a fluorogenic tag without any washing steps. Currently, further modifications are in progress to optimize the double ESPT systems with CyThioPhenol and further in-vivo applications. iii Dedicated to my parents, my family and Mehdi for their endless love, sacrifice, and support. iv ACKNOWLEDGEMENTS First, I would like to express my sincere gratitude and appreciation to my Ph.D. advisor, professor Babak Borhan for his endless patience, motivation, enthusiasm, and immense knowledge. Babak has been a tremendous mentor, being supportive and giving me the freedom to pursue various research projects. I want to thank him for encouraging my research and allowing me to grow as a scientist. I hope I’d be able to emulate his open-mindedness, creativity, and scientific rigor in my own career. He was more than just an advisor or professor to me, and numerous other students welcomed us at his home at Thanksgiving, Christmas, Persian, or Chinese New Year. These gatherings, along with some traditions unique to his lab, such as group camping in summer, fireworks on the 4th of July, and birthday cakes for everyone, helped me get through years far from my family. I cannot finish this part without mentioning how a great cook he is, being an expert in making the best Persian kabobs. Thanks for always being more than helpful; your advice on both research as well as on my career has been invaluable. I want to express my appreciation and gratitude to Dr. Chrysoula Vasileiou, one of the kindest people I know. She was a great help to me. I will never forget the hours I spent in her office asking questions or just talking about many different things. She always welcomed me warmly and advised me like an older sister. I will miss her and our lab gathering at her office. I want to thank our former lab member Dr. Wei Sheng who guided me throughout my Ph.D. studies and always had new fascinating ideas about our research, and I always enjoyed talking to him about v chemistry. I am also very thankful to Dr. Elizabeth Santos, Dr. Hadi Gholami, and Dr. Setare Tahmasebi Nick, who did not hesitate to teach me their skills and share their valuable knowledge and research experience. Our collaborators had a pivotal role in the success of many projects I would like to express my gratitude to Professor Geiger and his lab members, especially Dr. Alireza Ghanbarpour for his great work acquiring the crystal structures. Professor Gary Blanchard, and Dr. Wenjing Wang for their profitable share on the double ESPT project. I am very grateful to my committee members Professor Xuefei Huang, Professor Robert Maleczka and especially Professor James Geiger not only for their time and extreme patience, but for their intellectual contributions and support on my doctoral training and useful guidance and comments on my research projects. In addition, many multi-colored imaging microscopies would not have been possible without the guidance and expertise of Dr. Melinda Frame. A huge thank to my former and current lab mates and all my other friends, for their support, inspiration, and all fun we had during this journey. Finally, I want to deeply thank my parents, and sibling for their unconditional trust, endless patience, and selfless encouragement that let me explore new directions in life and seek my own destiny. I never can thank Mehdi enough; he is my best friend with an amazing unique personality, he made me happier than ever I imagined I could be. Without his positive sense of humor and sunny optimism it would be much more difficult. I am grateful to him because he has given up so much to make my career vi successful. He has shared this entire journey with me and has seen me through the entire ups and down of the graduate life, so it only seems right that I dedicate this milestone to him. vii TABLE OF CONTENTS LIST OF TABLES ................................................................................................ xii LIST OF FIGURES ............................................................................................ xvii KEY TO ABBREVIATIONS .............................................................................. xxvi CHAPTER I: A BRIEF OVERVIEW OF METHODS DEVELOPED FOR FLUORESCENT LABELING AND NEAR-INFRARED IMAGING TAGS…………1 I.1 Fluorescent proteins ........................................................................................ 1 I.1.1 Excited state proton transfer and the origin of intrinsic fluorescence in GFP…………. ...................................................................................................... .3 I.2 Far-red near-infrared synthetics fluorescent dyes ........................................... 8 I.3 Extrinsically fluorescent proteins and site-specific labeling methods ............. 10 REFERENCES.................................................................................................... 14 CHAPTER II: DEVELOPING FAR-RED/NEAR-INFRARED DYE-HCRBPII FLUORESCENT TAGS FOR NO-WASH BACKGROUND-FREE LIVE CELL IMAGING APPLICATIONS................................................................................. 29 II.1 Initial work towards developing hCRBPI-based fluorescent tags .................. 30 II.2 Desired features of a practical fluorescence imaging tag ............................. 34 II.3 Attempts to minimize non-specific fluorescent background .......................... 35 II.4 No-wash background-free fluorogenic imaging tag design ........................... 38 II.5 Spectroscopic properties of ThioPhenol in solution ..................................... 40 II.6 General protein host properties for this study ............................................... 43 II.7 Primary observation of dual fluorescence in hCRBPII/ThioPhenol ............. 44 II.8 The effect of rigidifying the chromophore through Y19W and A33W mutations on the absorption wavelength and ΦESPT ............................................................ 50 II.9 Exploring the effects of L117E and L117D mutations on the iminium pKa and ΦESPT………. ....................................................................................................... 52 II.9.1 Basic residues around the hydroxyl group ............................................ 54 II.9.2 Acidic residues around the hydroxyl group ........................................... 57 II.10 Investigating the effects of proton acceptor residues at positions A33 and F16 on the ΦESPT………………………………………………………………………………..58 viii II.11 Attempts to enhance the red-shifted emission quantum efficiency ............. 66 II.11.1 Through expansion of water-mediated hydrogen bonding network around the R58H residue .................................................................................... 66 II.11.1 A. T53 mutations ....................................................................... 69 II.11.1 B. S55 mutations ....................................................................... 71 II.11.1 C. Q38 mutations ...................................................................... 73 II.11.2 Enhancing the ESPT emission quantum efficiency through increasing the iminium pKa ................................................................................................... 78 II.11.2 A. L117 mutation ....................................................................... 81 II.11.2 B. Q4 and T51 mutation ............................................................ 88 II.11.2 C. K40 Mutation ........................................................................ 89 II.11.3 Through expanding water-mediated network around the hydroxyl group………. ....................................................................................................... 92 II.11.3 A. L77 mutation ......................................................................... 93 II.11.3 B. T29 mutation ......................................................................... 96 II.11.3 C. T53 mutation ........................................................................ 97 II-12 ThioPhenol/hCRBPII binding kinetics ...................................................... 101 II.13 Visualization of hCRBPII/ThioPhenol in mammalian cells ....................... 102 II.13.1 ThioPhenol/M3 successfully labels hCRBPII in HeLa cells ............ 103 II.14 Conclusion and future research directions ................................................ 105 REFERENCES.................................................................................................. 109 CHAPTER III: DOUBLE EXCITED-STATE PROTON TRANSFER PHENOMENON: DEVELOPING PROTEIN TAGS WITH APPLICATIONS IN NO- WASH FLUORESCENT IMAGING .................................................................. 122 III.1 Previous work toward developing large Stokes shift fluorescent protein-based photobases.. ..................................................................................................... 124 III.2 Designing hCRBPII/dye complexes that undergo double ESPT upon a single excitation….. ..................................................................................................... 130 III.2.1 General protein host properties required for a double ESPT system……........................................................................................................ 131 III.3 ThioPhenol-hCRBPII as a photobasic system .......................................... 132 III.4 Developing ThioPhenol-hCRBPII complexes showing both photoacidic and photobasic characteristics ................................................................................. 136 ix III.5 Attempts to increase the fluorescence quantum efficiency of DESPT derived emission…… ..................................................................................................... 140 III.5.1 Expanding the water-mediated hydrogen bonding network around the hydroxyl group .................................................................................................. 141 III.6. Application of the double ESPT systems as a fluorescent tag for live cells imaging……. ..................................................................................................... 148 III.7 Enhancing the double ESPT process with brighter chromophore cores .... 151 III.7.1 MR0… .............................................................................................. 152 III.7.1.A. Spectroscopic properties of MR0 in solution ........................ 152 III.7.1.B. Attempts to develop double ESPT process with MR0-hCRBPII complex……...................................................................................................... 159 III.7.2 MR1…. ............................................................................................. 169 III.7.2.A. Spectroscopic properties of MR1 in solution ........................ 170 III.7.2.B. Exploration about photoacidic and photobasic properties of MR1 complexes… .................................................................................................... .174 III.7.2.C. Developing protein-based double ESPT systems with MR1 186 III.8 ThioPhenol structure modification and its application in multicolor imaging……. ..................................................................................................... 192 III.8.1 Spectroscopic properties of CyThioPhenol in solution ................... 193 III.8.2 Developing CyThioPhenol photoacidic complexes ........................ 197 III.8.2. A. Kinetic measurements of CyThioPhenol binding hCRBPII Photoacid mutants ............................................................................................ 203 III.8.2. B. Visualization of hCRBPII/CyThioPhenol in mammalian cells…………..................................................................................................... 204 III.8.3 Developing the double ESPT systems with CyThioPhenol-hCRBPII complexes…. .................................................................................................... 209 III.8.4 Application of CyThioPhenol in multicolor no-wash live-cell confocal imaging……. ..................................................................................................... 220 III.8.4 A. Dual hCRBPII labeling expressed in HeLa cells ................... 222 III.9 Conclusion and future research plans ....................................................... 224 REFERENCES.................................................................................................. 228 CHAPTER IV: MATERIALS AND METHODS ................................................. 235 IV.1 Site-directed mutagenesis of hCRBPII and CRABPII ................................ 235 x IV.2 hCRBPII and CRABPII expression and purification in pET-17b expression plasmids…… ..................................................................................................... 245 IV.3 Protein characterization ............................................................................. 248 IV.3.1 UV/Vis and fluorescence spectroscopy .......................................... 248 IV.3.2 Extinction coefficient determination ................................................ 249 IV.3.3 pKa measurements of hCRBPII/chromophore complexes .............. 259 IV.3.4 hCRBPII/chromophore binding Kinetic measurements .................. 260 IV.3.4.A. Pseudo-first-order binding rate measurement ..................... 260 IV.3.4.B. Second-order binding rate measurement ............................ 261 IV.3.5. Absolute fluorescent quantum yield measurements ...................... 262 IV.4 Cloning to mammalian expression vectors ................................................ 262 IV.4.1 General cloning protocol .................................................................. 262 IV.4.2 Sequences of plasmids described in this thesis .............................. 264 IV.5 Mammalian cell culture and transfection ................................................... 271 IV.6 General methods for confocal imaging ...................................................... 272 IV.7 General synthetic procedures .................................................................... 274 IV.7.1 Synthesis of Thiophenol ................................................................. 275 IV.7.2 Synthesis of ThioPhenol-CF3 ......................................................... 278 IV.7.3 Synthesis of ThioPhenol-OMe2 ...................................................... 279 IV.7.4 Synthesis of MR0 ............................................................................ 280 IV.7.5 Synthesis of MR1 ............................................................................ 283 IV.7.6 Synthesis of Me-TR1 ....................................................................... 286 IV.7.7 Synthesis of CyThioPhenol ............................................................ 290 REFERENCES.................................................................................................. 296 xi LIST OF TABLES Table II-1. Spectroscopic characterization of ThioPhenol in various solvents. . 40 Table II-2. Spectroscopic characterization of ThioPhenol and derivatives ........ 43 Table II-3. Spectroscopic change as a result of mutating R58 in Q108K:K40L:T51V:T53S template. .................................................................... 46 Table II-4. Spectroscopic change upon the addition of Y19W and A33W mutations. ............................................................................................................................ 51 Table II-5. Spectroscopic change upon addition of L117E mutation. ................. 53 Table II-6. Spectroscopic properties of the protein mutants designed to increase the photoacidity of the bound ThioPhenol by introducing basic residues. ......... 56 Table II-7. Spectroscopic properties of the protein mutants designed to increase the photoacidity of the bound ThioPhenol by introducing acidic residues. ........ 58 Table II-8. Spectroscopic change upon mutation of A33 to histidine, serine and glutamic acid, and tyrosine. ................................................................................ 59 Table II-9. Spectroscopic change upon F16Y addition. ...................................... 61 Table II-10. Spectroscopic changes as a result of mutating F16. ....................... 63 Table II-11. Spectroscopic changes as a result of mutating R58, F16, and A33 residues to tyrosine. ............................................................................................ 65 Table II-12. Spectroscopic changes as a result of mutating T53 to histidine, tyrosine, and glutamic acid. ................................................................................ 69 Table II-13. Spectroscopic changes as a result of mutating T53. ....................... 70 Table II-14. Protein expression yields of hCRBPII mutants upon S55 mutation to histidine, tyrosine, and glutamic acid. ................................................................. 72 Table II-15. Spectroscopic properties of the PSB as a result of mutation of S55. ............................................................................................................................ 72 Table II-16. Spectroscopic properties of the PSB as a result of mutation of Q38. ............................................................................................................................ 74 xii Table II-17. Spectroscopic change as a result of mutation Q38 and T53 mutation to Glu................................................................................................................... 77 Table II-18. List of mutants that resulted in insoluble protein expression upon introduction of Glu resides in the proximity of iminium. ....................................... 81 Table II-19. Spectroscopic properties of protein mutants upon mutation of L117 in order to increase the iminium pKa. ...................................................................... 82 Table II-20. Spectroscopic properties as the result of L117M mutation. ............ 85 Table II-21. Spectroscopic properties of the SB as the result of Q4 mutation. ... 88 Table II-22. Spectroscopic change as the result of the introduction of T51M mutation. ............................................................................................................. 89 Table II-23. Spectroscopic properties as the result of K40 mutation. ................. 91 Table II-24. Spectroscopic change as the result of L77 mutations. .................... 93 Table II-25. Spectroscopic and pKa change as the result of L77Y introduction. . 94 Table II-26. Spectroscopic change as the result introduction of T29Y mutation. 96 Table II-27. Spectroscopic properties as the result of introduction of cysteine in positions T51 and T53. ....................................................................................... 97 Table II-28. Spectroscopic properties of ThioPhenol/M3 complex. ................. 103 Table III-1. Spectroscopic properties in complexation with M3. ....................... 132 Table III-2. Spectroscopic properties in complexation with M1. ....................... 136 Table III-3. Spectroscopic properties of ThioPhenol/M3 and M4 complexes. complexation with M1. ...................................................................................... 139 Table III-4. Spectroscopic properties upon addition of F16Y, A33H and T29Y 141 Table III-5. Spectroscopic properties as the result of substituting 58H with 58Y. .......................................................................................................................... 142 Table III-6. Spectroscopic properties of R58H containing mutants. ................. 145 Table III-7. Spectroscopic properties of ThioPhenol/M4 complex….…….........148 xiii Table III-8. Spectroscopic characterization of MR0 in various solvents. .......... 153 Table III-9. Spectroscopic features of MR0, FR0, and ThioPhenol in toluene and ethanol. ............................................................................................................. 154 Table III-10. Spectroscopic features of free aldehyde MR0 in water in acidic and basic pH. ........................................................................................................... 156 Table III-11. Spectroscopic characterization of MR0 and derivatives. .............. 158 Table III-12. Spectroscopic properties of MR0 Phenol-SB in PBS buffer. ........ 159 Table III-13. Spectroscopic properties of MR0/M3 and MR0/M4. ..................... 160 Table III-14. Spectroscopic properties of MR0/M3 and MR0/M4, and MR0/M5. .......................................................................................................................... 161 Table III-15. Spectroscopic properties change as the result of the addition of proton acceptor residues. ............................................................................................. 166 Table III-16. Spectroscopic characterization of MR1 in various solvents. ........ 170 Table III-17. Comparison of spectroscopic features of free aldehyde ThioPhenol and MR1 in ethanol. .......................................................................................... 172 Table III-18. Spectroscopic characterization of MR1 and derivatives. .............. 174 Table III-19. Comparison of the spectroscopic properties of FR1/M3 and MR1/M3 complexes. ........................................................................................................ 175 Table III-20. Spectroscopic changes as the result of addition of R58H mutation. .......................................................................................................................... 176 Table III-21. Spectroscopic properties of MR1 with high iminium and mutants 179 Table III-22. Spectroscopic properties of MR1 with high iminium and mutants. 180 Table III-23. Spectroscopic change as the result of L77Y and Q38Y mutations. .......................................................................................................................... 182 Table III-24. Comparison of the spectroscopic properties of FR1/M7 and MR1/M7 complexes. ........................................................................................................ 185 Table III-25. Comparison of spectroscopic properties of MR1 in complexation with M3 and M4 mutants. ......................................................................................... 186 xiv Table III-26. Spectroscopic change as the result of F77Y and Q38Y mutations. .......................................................................................................................... 188 Table III-27. Spectroscopic change as the result of the mutation of residues surrounding the MR1 hydroxyl group. ............................................................... 190 Table III-28. Spectroscopic characterization of CyThioPhenol in various solvents. .......................................................................................................................... 194 Table III-29. Spectroscopic characterization of CyThioPhenol and derivatives. .......................................................................................................................... 195 Table III-30. Spectroscopic change as the result of the addition of tyrosine and histidine residues around the hydroxyl group. .................................................. 198 Table III-31. Spectroscopic change as the result of the addition of L117C, T29Y, T51C and T53C mutations. ............................................................................... 201 Table III-32. Spectroscopic properties of CyThioPhenol/M6 complex compared with ThioPhenol/M3 complex. .......................................................................... 206 Table III-33. Spectroscopic properties of ThioPhenol and CyThioPhenol binding M3 mutant. ........................................................................................................ 209 Table III-34. Spectroscopic properties of mutants as the result of the introduction of V62E, I42E, and K40D mutations. ................................................................ 213 Table III-35. Spectroscopic properties of ThioPhenol and CyThioPhenol binding M4 mutant. ........................................................................................................ 214 Table III-36. Spectroscopic properties of mutants at positions 16, 20, 29, 33, 38, 58, and 77. ........................................................................................................ 216 Table III-37. Spectroscopic properties upon mutating the K40 residue. ........... 218 Table III-38. Spectroscopic properties of CyThioPhenol/M6 complex compared with CyThioPhenol/M3 complex. ..................................................................... 220 Table IV-1. PCR cycling conditions for site-directed mutagenesis. .................. 235 Table IV-2. FPLC Source 15Q method. ............................................................ 247 Table IV-3. FPLC SEC method. ....................................................................... 247 xv Table IV-4. Extinction coefficients of hCRBPII and CRABPII mutants. ............ 250 Table IV-5. PCR cycling conditions for cloning. ................................................ 263 xvi LIST OF FIGURES Figure I-1. A representative set of intrinsically fluorescent proteins and the corresponding chromophore structures responsible for fluorescence…………… 2 Figure I-2. Proposed mechanism for the maturation of the GFP chromophore... 4 Figure I-3. a. Equilibrium between the neutral (phenol) species and the anionic phenolate species of wt-GFP. b. Normalized fluorescence excitation (dashed line) and emission (solid line) spectra for wild-type GFP. Excitation at either peak (∼400 and 480 nm) leads to the characteristic green emission at 510 nm. ..................... 5 Figure I-4. Proposed water hydrogen bonding network for the excited state proton transfer in GFP. ..................................................................................................... 7 Figure I-5. Schematic self-labeling reactions of SNAP-tag and Halo Tag. POI: protein of interest. The red star represents the conjugated fluorophore………... 11 Figure I-6. Design of the PYP-tag mutant PYP3R and its fluorogenic probe, with a focus on electrostatic interactions and the pKa value of the leaving group.110 .... 12 Figure II-1. a. Crystal structure of Q108K:K40L-hCRBPII mutant complexed with all-trans-retinal. Key residues engineered to regulate the absorption wavelength of the bound retinylidene are highlighted (PDB 4EXZ). The scheme shows the iminium (PSB) formation between lysine 108 and retinal aldehyde. b. Protein solution of different hCRBPII mutants incubated with all-trans retinal………….. 31 Figure II-2. a. Structure of MCRA and its PSB formation with active Lysine 108 of hCRBPII. b. Spectroscopic properties of free aldehyde (blue) and PSB (red). .. 33 Figure II-3. Structures of Dapoxyl dye and ThioFluor………………………….... 37 Figure II-4. Non-specific fluorescent background originating from off-target iminium formation in labeled HeLa cells with 10 μM ThioFluor (incubated at 37 °C for 1 hour) expressing hCRBPII-EGFP-3NLS. NLS = nuclear localization sequence. ........................................................................................................... 37 Figure II-5. Structures of free aldehyde ThioPhenol, ThioPhenol-imine, ThioPhenol-iminium, and ThioPhenolate-iminium............................................ 39 Figure II-6. Spectroscopic properties of ThioPhenol in different solvents. a. UV- Vis and b. Fluorescence spectra of ThioPhenol…………………………………. 41 xvii Figure II-7. a. Schiff base and protonated Schiff base (PSB) of ThioPhenol with n-butyl amine in ethanol. b. Normalized spectra of ThioPhenol and derivatives in ethanol. Absorbance (left) and emission (right)……………………………... ........ 42 Figure II-8. Flexible docking of ThioPhenol in the crystal structure of Q108K:K40L:T51V:T53S:R58W-hCRBPII/ThioFluor. The distance between the hydroxyl group and the indole nitrogen of 58W side chain is 3.9 Å…………….. 45 Figure II-9. a. ThioPhenolate-PSB, the product of ESPT process, and normalized absorption/emission spectra of the iminium of Q108K:K40L:T51V:T53S:R58H/ThioPhenol complex. b. UV-Vis (left) and fluorescence (right) spectra of the same complex upon acidification of the solution. ............................................................................................................................ 47 Figure II-10. Formation of the ThioPhenolate-SB upon basification of the solution and the UV-Vis (left) and fluorescence (right) spectra of Q108K:K40L:T51V:T53S:R58H/ThioPhenol upon base titration……………….. 49 Figure II-11. Absorption and emission spectra of KLVS:R58W:Y19W:L117E:A33W/ThioPhenol complex upon PSB excitation. . 54 Figure II-12. Closest residues to ThioPhenol’s hydroxyl group in the crystal structure of Q108K:K40L:T51V:T53S:R58W:Y19W:L117D:Q4F/ThioPhenol complex………………………………………………………………………………... 55 Figure II-13. Hydrogen bonding network responsible for excited state proton transfer in LSS-mKate 1 and LSS-mKate 2……………………………………….. 57 Figure II-14. The crystal structure of Q108K:K40L:T51V:T53S:R58H:Y19W:A33Y/ThioPhenol complex with F16 mutated to tyrosine via mutagenesis in Pymol……………………………………. 60 Figure II-15. Schematic representation of the negative charge localization on the oxygen atom due to the electrostatic interaction with Tyr 16 side chain……….. 62 Figure II-16. a. Overlay of Q108K:K40L:T51V:T53S:R58W/ThioPhenol (magenta) and Q108K:K40L:T51V:T53S:R58H:Y19W:A33Y/ThioPhenol (green). b. The distance between the F16Y and the hydroxyl group. .............................. 64 Figure II-17. a. In silico modeling of R58H in the crystal structure of Q108K:K40L:T51V:T53S:R58W/ThioPhenol and the distances between S55, Q38, and T53S residues to 58H. b. T53S distances to R58H residue and ThioPhenol hydroxyl group…………………………………………………………. 67 xviii Figure II-18. Crystal structures of WT-hCRBPII bound with all-trans-retinol showing the internal hydrogen bonding network among the T51 and T53 side chain residues and retinol…………………………………………………………………... 68 Figure II-19. Water mediated hydrogen bonding between Q38 and Q128 in the crystal structure of hCRBPII-Q108K:K40L/retinal PDB 4EXZ……………………. 73 Figure II-20. a. Overlay of Q108K:K40L:T51V:T53S:R58W/ThioPhenol (green) and Q108K:K40L/retinal (pink). b. Water network between T53S, Q38, and Q128 in Q108K:K40L:T51V:T53S:R58W/ThioPhenol crystal structure. ..................... 75 Figure II-21. a. UV-Vis (left) and fluorescence (right) spectra of Q108K:K40L:T51V:T53S:R58H:A33H:F16Y:Q38E/ThioPhenol upon acid titration. b. Excitation of the shoulder indicated by the black arrow results in the same emission as PSB excitation…………………………………………………... 76 Figure II-22. a. An overlay of crystal structures of Q108K:K40L:T51V:T53S:R58W:Y19W:L117D:Q4F/ThioPhenol (purple) and ThioFluor (green) showing cis and trans iminium conformation, respectively. b. The distance between L117D residue and the iminium nitrogen atom for ThioFluor complex (left) and ThioPhenol (right)……………………………………………….79 Figure II- 23. Highlighted residues mutated to glutamic acid in order to interact with the iminium to increase its pKa…………………………………………………. 80 Figure II-24. UV-Vis (left) and fluorescence (right) spectra of Q108K:K40L:T51V:T53S:R58H:Y19W:A33H:F16Y/ThioPhenol (red) and Q108K:K40L:T51V:T53S:R58H:Y19W:A33H:F16Y:L117T/ThioPhenol (blue). . 84 Figure II-25. a. Water mediated hydrogen bonding between Q4 and the iminium, and the distance between L117C and the iminium nitrogen atom in the crystal structure of Q108K:K40L:T51V:T53S:R58H:Y19W:A33H:F16Y:L117C/ThioPhenol complex. b. The different trajectory of chromophore shown upon the overlay of Q108K:K40L:T51V:T53S:R58H:Y19W:A33H:F16Y:L117C/ThioPhenol (green) and Q108K:K40L:T51V:T53S:R58W:Y19W:L117D: Q4F/ThioPhenol (purple) with F16 residue mutated to Try in the latter (shown in grey)……………………. 87 Figure II-26. The distance between the K40L residue and the iminium nitrogen atom in the crystal structure of Q108K:K40L:T51V:T53S:R58H:Y19W:A33H:F16Y:L117C/ThioPhenol. .......... 90 Figure II-27. a. Cartoon representation of Q108K:K40L:T51V:T53S:R58H:Y19W:A33H:F16Y:L117C/Thiophenol complex xix with T29 and L77 residues highlighted. b. Detailed hydrogen bonding network surrounding T29, A33H, R58H and the hydroxyl group in the same crystal……. 95 Figure II-28. a and b. The absorption and emission spectra of Q108K:K40L:F16Y:Y19W:T29Y:A33H:T51C:T53S:R58H:L117C/ThioPhenol complex. The shoulder corresponds to PSB-Phenol complex since excitation of both results to the same emission. c. The acid titration and d. the base titration of the same complex……………………………………………………………………. 99 Figure II-29. Comparison of absorption (left) and emission (right) of M1 (T51VT:53S), M2 (T51C:T53S), M3 (T51V:T53C), and M4 (T51M:T53S) protein/ThioPhenol complexes……………………………………………………. 100 Figure II-30. Rate of ThioPhenol/M3 PSB formation, fitted to 2nd order kinetics with 20 μM protein and 10 μM Thiophenol. Plotted is the concentration of free chromophore vs. time. ...................................................................................... 101 Figure II-31. Spectroscopic properties of Q108K:K40L:F16Y:Y19W:T29Y:A33H:T51V:T53C:R58H:L117C mutant with ThioPhenol including UV-Vis and fluorescence spectra (left) and pKa titration (right)…………………………………………………………………………………. 102 Figure II-32. Maps of the EGFP-hCRBPII-SP fusion constructs. SP: signaling peptides. SP = 3×NLS (nuclear localization sequence), NES (nuclear export sequence), and CAAX (prenylation tag). .......................................................... 103 Figure II-33. Confocal imaging of labeled HeLa cells expressing EGFP-hCRBPII- 3NLS. NLS = nuclear localization sequence. Cells were stained with 10 µM ThioPhenol and incubated at 37 °C for 1 h and 30 min. Cells were not washed before imaging………………………………………………………………………. 107 Figure II-34. Confocal imaging of labeled HeLa cells expressing EGFP-hCRBPII- NES. NES = nuclear export localization sequence. Cells were stained with 10 µM ThioPhenol and incubated at 37 °C for 1 h and 30 min. Cells were not washed before imaging………………………………………………………………………. .108 Figure III-1. Model for a large Stoke shift (LSS) vs. a standard fluorescence system. Large Stokes shift red-shifted emission results via conversion of the excited state species A* to a bathochromically distinct molecule B*………….. 124 Figure III-2. a. FR0 structure and formation of FR0-SB and FR0-PSB. b. UV-Vis (left) and fluorescence (right) spectra of FR0-SB (blue) and FR0-PSB (red) in ethanol……………………………………………………………………………….. 125 xx Figure III-3. a. Formation of the imine and iminium of ThioFluor and FR1 in ethanol. b. Spectroscopic properties of FR0 (blue), FR1 (red), and ThioFluor (green) in ethanol. UV-Vis (left) and fluorescence spectra upon SB excitation (right). ................................................................................................................ 127 Figure III-4. UV-Vis and fluorescence spectra of M3/ThioFluor complex (394 nm excitation), exhibiting ESPT emission (left) vs. UV-Vis and fluorescence spectra of M1/ThioFluor complex (380 nm excitation) leads to SB emission at 474 nm (right). The spectra have been measured in PBS buffer at neutral pH (7.2)………….. 128 Figure III-5. M3/FR1 imaging in live HeLa cells. NES = nuclear export sequence. Cells were stained with 500 nM FR1 and incubated at 37 °C for 1 min. Cells were washed three times with DPBS before imaging. Scale bar, 10 𝜇m……………. 129 Figure III-6. a. Formation of ThioPhenolate-PSB complex through a double ESPT process. b. The internal charge transfer resulted from the ThioPhenolate-PSB complex, the product of the double ESPT process……………………………….131 Figure III-7. UV-Vis, emission spectra of Q108K:K40E:T53A:R58L:Q38F:Q4F/ThioPhenol complex……………………. 133 Figure III-8. a. UV-Vis (left) and fluorescence (right) spectra of Q108K:K40E:T53A:R58L:Q38F:Q4F/ThioPhenol complex upon acidification of the solution. b. UV-Vis (left) and fluorescence (right) spectra of Q108K:K40D:T53A:R58L:Q38F:Q4F/ThioFluor complex upon acidification of the solution. ............................................................................................................. 134 Figure III-9. UV-Vis (left) and fluorescence (right) spectra of Q108K:K40E:T53A:R58L:Q38F:Q4F/ThioPhenol complex upon basification of the solution…………………………………………………………………………... 135 Figure III-10. Normalized absorption and emission spectra of Q108K:K40E:T53A:R58H:Q38F:Q4F/ThioPhenol complex, when excited at 370 nm at neutral pH (~7.2)……………………………………………………………... 137 Figure III-11. Schematic representations of single and double ESPT processes. The starting complex is different, while the product is the same. ..................... 138 Figure III-12. a. UV-Vis spectra of Q108K:K40E:T53A:R58H:Q38F:Q4F/ThioPhenol complex upon acid titration of the sample. b. Fluorescence spectra of the same complex upon SB excitation at neutral pH and PSB excitation in acidic pH. ..................................................... 139 xxi Figure III-13. UV-Vis spectra of Q108K:K40E:T53A:R58H:Q38F:Q4F/ThioPhenol complex upon base titration (left). Fluorescence spectra of the same complex upon SB excitation in different pH. ............................................................................. 140 Figure III-14. The crystal structure of Q108K:K40E:T53A:R58W:Q38F:Q4F:Y19W/ThioFlour and the closest residues to the nitrogen atom of the dimethyl amino moiety of ThioFluor………………. 144 Figure III-15. Products of single ESPT and double ESPT processes. Absorption and emission spectrum of Q108K:K40E:T53A:Q38F:Q4F:R58H:L77M/ThioPhenol upon SB excitation at 370 nm……………………………………………………………………………….. 146 Figure III-16. Absorption (left) and emission spectra (right) of Q108K:K40E:T53A:Q38F:Q4F:R58H:F16Y:M20Y/ThioPhenol complex. Upon excitation of SB (blue line) and excitation of the PSB (red line). DESPT= Double ESPT. SESPT= Single ESPT. .......................................................................... 147 Figure III-17. Confocal imaging of labeled HeLa cells expressing EGFP-hCRBPII- whole cell. Cells were stained with 10 µM ThioPhenol and incubated at 37 °C for 5 min. Cells were not washed before imaging……………………………………. 150 Figure III-18. Structures of ThioPhenol, MR0, and FR0.................................. 152 Figure III-19. Spectroscopic properties of MR0 in different solvents. a. UV-Vis and b. Fluorescence spectra. .................................................................................. 154 Figure III-20. UV-Vis spectra (left) and fluorescence spectra (right) of free aldehyde MR0 upon acidification and basification of the sample. Measurements were done in water………………………………………………………………….. 155 Figure III-21. a. Schiff base and protonated Schiff base (PSB) of MR0 with n-butyl amine in ethanol. b. Absorbance (left) and emission (right) spectra of MR0 and derivatives: Phenol-SB, Phenol-PSB, and Phenolate-PSB. ............................. 157 Figure III-22. Absorption (left) and emission (right) spectra of M3 (a), M4 (b), and M5 (c) upon basification of their solutions. ....................................................... 162 Figure III-23. Formation of the hydrolyzed Phenolate-SB complex produced upon basification of the protein solution. .................................................................... 163 Figure III-24. Absorption (blue line) and emission (red line) of Q108K:K40L:T51V:T53S:R58H:F16Y:Y19W:A33H:L117E/MR0 complex at neutral (left) and acidic (right) pH…………………………………………………. 164 xxii Figure III-25. The schematic representation of single and double ESPT processes and their photophysical properties………………………………………………... 167 Figure III-26. Absorption and emission spectra of Q108K:K40E:T53A:Q38F:Q4F:R58H:F16Y/MR0 complex. The blue-shifted shoulder corresponds to Phenol-PSB, or Phenolate-SB produced upon single ESPT, and the emission at 560 nm results from Phenolate-PSB complex produced upon the double ESPT process……………………………………………………. 168 Figure III-27. Structures of FR1 and MR1……………………………………….. 170 Figure III-28. Spectroscopic properties of MR1 in different solvents. a. UV-Vis and b. Fluorescence spectra. .................................................................................. 171 Figure III-29. a. Schiff base and protonated Schiff base (PSB) of MR1 with n- butylamine in ethanol. b. Absorbance (left) and emission (right) spectra of MR1 and derivatives: Phenol-SB, Phenol-PSB, and Phenolate-PSB…………….......173 Figure III-30. The normalized absorption (blue line) and emission (red line) spectra of MR1/M3 complex…………………………………………………………………. 175 Figure III-31. The absorption and emission spectra of Q108K:K40L:T51V:T53S:R58H/MR1 complex a. upon SB excitation in neutral pH 7.2 and b. PSB excitation in acidic pH 5.4………………………………………... 177 Figure III-32. Proposed photoswitching cycle of Q108K:K40L:T51V:T53S:R58H/MR1 complex…………………………………... 178 Figure III-33. Flexible docking of MR1 in the crystal structure of Q108K:K40L:T51V:T53S:R58W-hCRBPII/ThioFluor. MR1 is shown in purple, and ThioFluor is shown in cyan………………………………………………………... 181 Figure III-34. Absorption (blue line) and emission (red line) spectra of Q108K:K40L:T51V:T53S:R58H:Y19W:A33H:F16Y: L117C:L77Y/MR1 complex upon excitation of a. SB at 364 nm and b. PSB at 470 nm in neutral pH……... 183 Figure III-35. Absorption (left) and emission (right) spectra of Q108K:K40L:T51V:T53S:R58H:Y19W:A33H:F16Y: L117C:L77Y/MR1 upon acid and base titrations…………………………………………………………………... 184 Figure III-36. The absorption and emission spectra of Q108K:K40E:T53A:Q38F:Q4F:R58H/MR1 complex measured at neutral pH...187 xxiii Figure III-37. The absorption and emission spectra of Q108K:K40E:T53A:Q38F:Q4F:R58H:F16Y:Q38Y/MR1 complex measured at neutral pH. ........................................................................................................ 188 Figure III-38. Docked MR1 in the crystal structure of Q108K:K40L:T51V:T53S:R58W-hCRBPII/ThioFluor, and the closest residues around the hydroxyl group…………………………………………………………. 189 Figure III-39. a. Structures of ThioPhenol and CyThioPhenol. b. Absorbance (left) and emission (right) spectra of CyThioPhenol in different solvents…... .. 193 Figure III-40. Absorbance (left) and emission (right) spectra of CyThioPhenol and …derivatives: Phenol-SB, Phenol-PSB, and Phenolate-SB……………………. 195 Figure III-41. Schematic comparison of ICT systems in Phenolate-PSB and Phenolate-SB complexes of CyThioPhenol…………………………………….. 197 Figure III-42. a. The absorption and b. the emission spectra of KLVSH is equal to Q108K:K40L:T51V:T53S:R58H:F16Y:L77Y/CyThioPhenol complex measured in neutral pH 7.3. c, d. The absorption and emission spectra of the same complex screened upon acidification pH 5.8 and basification pH 8.3 of the solution….. 199 Figure III-43. The absorption spectra of Q108K:K40L:T51V:T53C:R58H:Y19W:F16Y:T29Y:L77Y:L117C/CyThioPhenol complex recorded upon basifying the solution from pH 7.3 to pH 9.5. ............. 202 Figure III-44. Rate of CyThioPhenol/M6 PSB formation fitted to second-order kinetics with 20 μM protein and 10 μM CyThioPhenol. Plotted is the concentration of free chromophore vs. time………………………………………………………. 204 Figure III-45. Spectroscopic properties of Q108K:K40L:T51V:T53C:R58H:Y19W:A33H:F16Y:L77Y:L117C mutant with CyThioPhenol including UV-Vis and fluorescence spectra (left) and the pKa titration (right)……………………………………………………………………….. . 205 Figure III-46. Schematic map of EGFP-hCRBPII-NES fusion construct. NES: nuclear export sequence………………………………………………………….... 207 Figure III-47. Confocal imaging of labeled HeLa cells expressing EGFP-hCRBPII- NES. NES: nuclear export sequence. Cells were stained with 5 µM CyThioPhenol and incubated at 37 °C overnight. Cells were not washed before imaging. ...... 208 xxiv Figure III-48. The absorption and emission spectra of a. ThioPhenol/M3 (red lines) and b. the absorption and emission spectra of CyThioPhenol/M3 complex (blue lines)......................................................................................................... 210 Figure III-49. The absorption and emission spectra of Q108K:K40L:T53A:Q38F:Q4F:R58L/CyThioPhenol complex measured in neutral pH...................................................................................................................... 211 Figure III-50. The crystal structure of Q108K:K40E:T53A:R58W:Q38F:Q4F:Y19W/ThioFlour and highlighted V62, I42, and K40E residues. ........................................................................................... 212 Figure III-51. Absorption and emission spectra of Q108K:K40E:T53A:Q38F:Q4F:R58H/CyThioPhenol complex measured in neutral pH. ........................................................................................................ 214 Figure III-52. The absorption and emission spectra of CyThioPhenol upon binding a. Q108K:K40E:T53A:Q38F:Q4F:R58H:A33H and b. Q108K:K40E:Q4F:A33H:Q38F:T53A:R58H:L77Y mutants…………………….. 217 Figure III-53. The absorption and emission spectra of Q108K:K40D:T53A:Q38F:Q4F:R58H/CyThioPhenol complex upon SB excitation……………………………………………………………………………... 219 Figure III-54. a. M3/CyThioPhenol complex pKa titration. b. The absorption (left) and emission (right) spectra of M3 (blue) and M6 (red) mutants upon binding CyThioPhenol measured at pH 7.2………………………………………………. 221 Figure III-55. Schematic map of EGFP-M6-hCRBPII-NES and EGFP-M3- hCRBPII-3NLS fusion constructs. NLS: nuclear localization sequence and NES: nuclear export sequence. .................................................................................. 222 Figure III-56. Confocal imaging of labeled HeLa cells expressing EGFP-hCRBPII- 3NLS. NLS = nuclear localization sequence. Cells were stained with 5 mM CyThioPhenol and incubated at 37 °C for 5 min. Cells were not washed before imaging.............................................................................................................. 226 Figure III-57. Confocal imaging of labeled HeLa cells expressing EGFP-hCRBPII- 3NLS and EGFP-hCRBPII-NES. NLS= nuclear localization sequence. NES= nuclear export sequence. Cells were stained with 5 µM CyThioPhenol and incubated at 37 °C for overnight and another 5 µM CyThioPhenol incubated at 37 °C for 5 minutes. Cells were not washed before imaging…………………….... 227 xxv KEY TO ABBREVIATIONS A alanine, Ala C cysteine, Cys D aspartic acid, Asp E glutamic acid, Glu F phenylalanine, Phe G glycine, Gly H histidine, His I isoleucine, Ile K lysine, Lys L leucine, Leu M methionine, Met N asparagine, Asn P proline, Pro Q glutamine, Gln R arginine, Arg S serine, Ser T threonine, Thr V valine, Val W tryptophan, Trp Y tyrosine, Tyr xxvi Å angstrom, 10-10 meter Abs, λabs absorbance, absorption wavelength maximum ACQ aggregation-caused quenching AIE aggregation-induced emission AIEgen aggregation-induced emission luminogen/fluorogen aq. Aqueous BFP blue fluorescent protein BODIPY boron dipyrromethene BP bandpass (filter) ℃ degree Celsius CAAX prenylation tag CFP cyan fluorescent protein cm centimeter cm-1 wavenumber CP chromoprotein CRABPII cellular retinoic acid binding protein II CuAAC CuI-catalyzed alkyne-azide cycloaddition Da Dalton DESPT double excited state proton transfer DIBAL diisobutylaluminium hydride DIC differential interference contrast DMEM Dulbecco’s Modified Eagle’s Medium xxvii DMSO dimethyl sulfoxide DPBS Dulbecco’s phosphate buffered saline EDG electron donating group eDHFR Escherichia coli dihydrofolate reductase EGFP enhanced green fluorescent protein Em, λem emission, emission wavelength maximum equiv equivalent ESCT excited state charge transfer ESIPT (ESiPT) excited state intramolecular proton transfer ESPT excited state proton transfer EWG electron withdrawing group Ex, λex excitation wavelength FABP fatty acid binding protein FAP fluorogen-activating proteins FBS fetal bovine serum FL fluorescence FlAsH fluorescein arsenical hairpin binder FP fluorescent protein FPLC fast protein liquid chromatography FRET Förster resonance energy transfer FRFP far-red fluorescent protein g gram xxviii GFP green fluorescent protein h hour hAGT human O6-alkylguanine-DNA alkyl transferase HBA H-bonding acceptor HBD H-bonding donor HBR 4-hydroxybenzylidene-rhodanine hCRBPII human Cellular Retinol Binding Protein II HOMO highest occupied molecular orbital Hz hertz IC internal conversion ICG indocyanine green ICT intramolecular charge transfer IDT Integrated DNA Technologies IEDDA Inverse-Electron-Demand Diels-Alder cycloaddition iLBP intracellular lipid binding protein IPTG isopropyl β-D-1-thiogalactopyranoside IR infrared ISC intersystem crossing K kelvin k kinetic rate constant LB lysogeny broth LP longpass (filter) xxix LpIA lipoic acid ligase LSS long/large Stokes shift LUMO lowest unoccupied molecular orbital M molar mBeRFP monomer blue light-excited RFP MBP maltose binding protein MCRA merocyanine retinal aldehyde mg milligram min minute mmol millimole mol mole MS molecular sieve N.D. not determined N.O. not observed NBD nitrobenzoxadiazole NES nuclear export sequence NIR near-infrared NLS nuclear localization sequence nm nanometer nM nanomolar O/N overnight OD optical density xxx OFP orange fluorescent protein p-HBI 4-(p-hydroxy-benzylidene)-5-imidazolinone PA-FP photoactivatable fluorescent protein PBS phosphate-buffered saline PCR polymerase chain reaction PDB protein data bank PeT photoinduced electron transfer PHY phytochrome PLICT planarized intramolecular charge transfer POI protein of interest PPI protein-protein interaction PSB protonated Schiff base PSFP photoswitchable FP PYP photoactive yellow protein qABP quenched activity-based probe QY quantum yield ReAsH resofurin arsenical hairpin binder RFP red fluorescent protein ROI region of interest SB Schiff base SBR signal-to-background ratio sec second xxxi SEC size-exclusion chromatography SNAr nucleophilic aromatic substitution SNR signal-to-noise ratio SPAAC strain-promoted alkyne-azide cycloaddition SQ Source 15Q SS Stokes shift T temperature t time t1/2 maturation half-life TB Terrific broth TBET through-bond energy transfer THF tetrahydrofuran TICT twisted intramolecular charge transfer TMP trimethoprim UV ultraviolet Vis visible wtGFP wild-type green fluorescent protein xs excess Y-FAST Yellow Fluorescence-Activating and absorption-Shifting Tag YFP yellow fluorescent protein ε extinction coefficient μg microgram xxxii μm micrometer μM micromolar 𝝂 frequency Φ quantum yield xxxiii CHAPTER I: A BRIEF OVERVIEW OF METHODS DEVELOPED FOR FLUORESCENT LABELING AND NEAR-INFRARED IMAGING TAGS Revolutionary advances in science and technology have greatly enhanced our lives. The development of fluorescent proteins (FPs) and their subsequent use in fluorescent imaging is an excellent example of basic science leading to practical biotechnological and medical applications. Fluorescence-based assays are crucial tools, which enable studying detailed molecular mechanisms, including protein- protein interactions, enzymatic activity, conformational changes, and protein localization.1-9 I.1 Fluorescent proteins Fluorescent proteins (FPs) have empowered researchers to visualize inside living cells, examine tissues and subcellular components, and track biological and cellular events with an unprecedented level of resolution and sensitivity.10-12 Engineered fluorescent proteins from various species have expanded the color palette from blue to red and far-red spectral regions.13 Notably, engineered fluorescent proteins from bacterial phytochromes emit around near-infrared (NIR) wavelengths (Figure I-1).14-16 The green fluorescent protein (GFP) and its color-shifted genetic variants have played an indispensable role in many live-cell imaging experiments. FPs are genetic labels and can be fused to the target proteins without the need for exogenous labeling agents or permeabilization procedures.17,18 They are 1 expressed in a 1:1 ratio (if fused to one FP), which leads to extremely high labeling specificity.19,20 O N O N N N O N N O Citrine N N O N ECFP λex = 516 nm H λex = 433 nm λem = 529 nm R λem = 475 nm O mCherry λex = 587 nm λem = 610 nm ew e rry rry nana mOr ato erry mGr mGr m e EBF ECF EGF Citrin gerin P P P neyd ange tdTom mStr mCh ape1 spbe ape2 mPlu mBa mTan awbe mRa mHo O N O N N N NH EBFP N N OH λex = 383 nm O λem = 445 nm O mOrange O N λex = 548 nm N λem = 562 nm EGFP O λex = 484 nm λem = 507 nm Figure I-1. A representative set of intrinsically fluorescent proteins and the corresponding chromophore structures responsible for fluorescence. In recent years, red-shifted FPs have become more desired in bioimaging applications because cells are more transparent to far-red/NIR light while effectively absorb in shorter wavelengths.21-24 In addition, cellular autofluorescence decreases as the excitation wavelengths increases.24,25 Imaging 2 with fluorophores that have red-shifted excitation maxima can also benefit sample health. The light of a longer wavelength is less phototoxic to cells and will enable longer acquisition times.26,27 However, known naturally occurring red fluorescent proteins (RFPs), such as DsRed,28 and eqFP611,29 tend to form obligate oligomers, and their usefulness as molecular fusion tags in in-vivo model systems are limited.30,31 Using directed evolution, significant effort has been put towards monomerizing the RFPs mentioned above. It is noteworthy that all the mFruits, including the most widely used mCherry,32 tandem dimer tdTomato,32 and TagRFP,33 are derived from naturally tetrameric proteins. Random mutagenesis has successfully yielded several far-red (λem >630 nm) monomeric RFPs such as mPlum,29 mKate2,34 and mNeptune.35 Despite many advantageous features, FPs do not always exhibit large Stokes shifts and are oxygen-dependent. In addition, some have residual tendencies to oligomerize and aggregate, which can cause localization defects that are detrimental to certain applications.36-38 I.1.1 Excited state proton transfer and the origin of intrinsic fluorescence in GFP As discussed above, FPs do not require an accessory cofactor, external enzymes, or an exogenous substrate to fluoresce. Particularly in green fluorescent protein (GFP), the light-emitting molecular unit, the chromophore, is p- hydroxybenzylidene-2,3-dimethylimidazolinone (HBDI) in its anionic form (Figure 3 I-2). The maturation of the embedded chromophore is accomplished through an autocatalytic process. The most accepted mechanism for the maturation of p-HBI, which was acquired based on several crystallographic studies, is depicted in Figure 1-2.39 As shown below, formation of the chromophore happens in three subsequent steps: 1) internal nucleophilic attack of Gly67 amide nitrogen to the carbonyl carbon of Ser65 that results in the formation of a five-membered imidazolone ring, 2) dehydration of the hemi-aminal to form an imidazolin-5-one intermediate, (3) oxidation of Tyr66 by molecular oxygen that completes the conjugation of the ring system (Figure 1-2).40,41 HO Tyr66 Gly67 O O H N N N N H H Folding H O O HN O O HO OH N Ser65 H OH Cyclization O O H2O2 O2 N N N HN O -H2O OH O HO HO N N Mature H H Chromophore OH OH p-HBI Figure I-2. Proposed mechanism for the maturation of the GFP chromophore. 4 However, neither unfolded GFP, nor the naked chromophore (p-HBI), are fluorescent proving that the stable protein structure is a requirement for efficient fluorescence. Notably, GFP is extremely resistant to protease enzymes and remains stable under very harsh conditions making it exceptionally suitable as a fusion tag in both prokaryotic and eukaryotic organisms.42-44 a. O O N N H+ N N O O HO O A N B N H H OH OH b. Band A 1 - - 1 Band B Figure I-3. a. Equilibrium between the neutral (phenol) species and the anionic phenolate species of wt-GFP. b. Normalized fluorescence excitation (dashed line) and emission (solid line) spectra for wild-type GFP. Excitation at either peak (∼400 and 480 nm) leads to the characteristic green emission at 510 nm. 5 Although several GFP homologs have been isolated from marine organisms, the original GFP isolated from Aequorea victoria shows unique and complex excitation/emission spectra. The absorption spectrum of the wild-type green fluorescent protein (wt-GFP) contains two peaks: at 395 nm, referred to as band A, and at 475 nm, referred to as band B.45 Excitation of either of the two absorption bands leads to emission at 510 nm. This observation suggests an equilibrium exists between two distinct chromophore states within the protein with similar emission wavelengths (Figure 1-3). Boxer and co-workers discovered that the apparent large shift of the emission to longer wavelengths after excitation at band A is due to the deprotonation of the neutral chromophore (phenolate) induced by the excited-state proton transfer (ESPT) process occurring via a hydrogen-bonded network.46-48 They showed that the light-driven conversion between the neutral (A) and anionic (B) form of the chromophore passes through the intermediate state I (Figure I-4). Moreover, X-ray crystallographic data provided by Remingtion, Tsien, and co-workers confirmed a hydrogen-bonding network that links the chromophore hydroxyl group and E222. It is proposed that photoexcited A-form (A*) transfers a proton to E222 via a water molecule and S205 (Figure I-4).45 Quantum-classical calculations support these findings and reveal that the phenolic oxygen acidity increases and its pKa drops by several units upon photoexcitation, which in correlation with the heterocyclic ring, provides an emissive push-pull system.45,46,49 6 GFP is an exemplar of a naturally occurring biological system in which the ESPT phenomenon strongly influences its fluorescence properties.39,48,50 Notably, the dual excitation behavior of GFP have found applications in designing active biosensors of various cellular phenomena.51 Thr203 O O Thr203 O N R N R H O N H N N N O R O R N H Ser65 N Ser65 His148 A H B O O H His148 H O H O H H O H R H H O OH H O O H O H Asn146 R O O O R Glu222 Asn146 Glu222 O Ser205 R Ser205 Thr203 Thr203 λabs = 396 nm λabs = 475 nm λem = 508 nm λem = 503 nm ESPT Thr203 O O N R H N N O R N I Ser65 His148 O H H O H O R H O O H H Asn146 O O R Glu222 Ser205 Thr203 Figure I-4. Proposed water hydrogen bonding network for the excited state proton transfer in GFP. The ESPT phenomenon has been further adapted to engineer FPs in order to extend the Stokes Shift, such as mKeima, LSSmKates, mBeRFP, CyOFP1, Sandercyanin, and TagRFP675, to name a few.37,52-57 The chromophore environment of this class of proteins shares noticeable structure similarities; as in 7 GFP, the hydroxyl group of the chromophore forms a hydrogen-bonding network with active site neighboring residues. The web of hydrogen bonding both stabilizes the neutral form of the chromophore in the ground state and facilitates deprotonation of the tyrosine hydroxyl group through ESPT process in the excited state. I.2 Far-red near-infrared synthetics fluorescent dyes Recently, noninvasive optical imaging in the far-red/near infra-red (NIR) region has attracted significant attention due to deeper penetration (approximately 5-20 mm) of biological tissues, reduced autofluorescence background, and improved signal-to-noise ratio.22,58-62 Lately, organic fluorescent dyes have become increasingly attractive from a practical perspective because their photophysical properties are well-tunable by various structural modifications.63-65 Additionally, their small size can be of great benefit, which ensures low interference to the function of the labeled protein. Some organic dyes also offer higher photostability and brightness.66,67 Red- and NIR- emitting organic fluorophores belong to various dye families, such as cyanines,68,69 Si-rhodamines,70,71 and BODIPY72,73 derivatives. Most red- emitting fluorophores have extensive p-electron conjugation, usually containing large polycyclic aromatic hydrocarbons. Some other dyes have a push-pull type electronic structure that renders red/far-red fluorescence when placed in a polar milieu and favors positive solvatochromism. 8 However, oftentimes structural modifications are needed to improve their permeability and solubility. For example, because of intermolecular stacking or attractive dipole-dipole interactions, many organic dyes are prone to form aggregates in aqueous environments, resulting in drastic self-quenching, aggregation-caused quenching (ACQ),38,74-76 and can significantly impede their applications in many biological systems.77,78 Nonetheless, the AIEgen materials, known to have aggregation-induced emission (AIE), are weakly emissive or non-emissive in dilute solutions but become highly emissive in aggregated or solid form and can have potential use in some systems.79,80 Well-known cyanine dyes also have small Stokes shifts (typically less than 35 nm), which leads to severe self-quenching81 and cause poor Signal-to-Noise Ratio (SNR) in imaging.82 Organic fluorescent dyes do not need maturation half-time that can vary from minutes to hours for FPs; however, the unbound fluorophore and/or nonspecifically bound fluorophores can cause severe fluorescent background. consequently, the use of non-fluorogenic fluorophores that are always “ON” (here defined as always being fluorescent) requires extensive washing steps to remove excess dye prior to imaging.83 An alternative approach to overcome this problem is to use fluorogenic dyes. These fluorochromes fluoresce upon binding to the target and are particularly useful for bioimaging.84,85 Solvatochromicity,86 pH sensitivity,87 photoinduced electron transfer (PeT),88 and other dark state quenching 9 mechanisms89,90 are among the current methods employed to produce fluorogenic dyes in order to avoid the need for washing steps. For example, quenched activity-based probes (qABPs)- they are basically small molecule reporters of enzymatic activity- are commonly designed to compose a ligand-tethered fluorophore and a quencher via a certain linkage that can be cleaved by key catalytic residues of the enzyme with high selectivity. This enzymatic activity-dependent cleavage leads to the release of the quencher moiety and subsequent restoration of reporter fluorescence.90-92 Tetrazine-derived fluorogens are another example that can put the fluorophores into quiescence before target binding. However, it requires the expression of non-canonical amino acids chemically modified with olefinic side chains to facilitate the fluorescence activation via sequential inverse electron demand Diels-Alder (IEDDA) reactions and dinitrogen repulsion.93,94 Despite laborious design efforts to customize quenching mechanisms for a given fluorophore, this fluorogenic approach sometimes suffers the drawbacks such as modest fluorescent enhancement or incomplete fluorescence quenching. I.3 Extrinsically fluorescent proteins and site-specific labeling methods The conjugation of small dyes with genetically encodable protein/peptide tags has led to the development of flexible labeling systems that can be tailored ad infinitum. These site-selective chemical labeling systems include self-labeling enzymes SNAP/CLIP-tag and HaloTag (Figure 1-5),95,96 short peptide tags that bind biarsenical FlAsH and ReAsH97,98, PYP-based fluorogenic protein tags 10 (Figure 1-6),99,100 protein affinity ligands (Y-FAST, TMP, and FAP),9,101-103 and bioorthogonal tags namely copper-catalyzed alkyne-azide cyclization (CuAAC), strain-promoted alkyne-azide cycloaddition (SPAAC), Staudinger ligation, and other copper-free click reactions.91,104-106 SNAP Tag POI S POI S O N N O N N NH2 N H NH N N NH2 H Halo Tag O O POI O POI O H O N O O Cl Cl O NH Figure I-5. Schematic self-labeling reactions of SNAP-tag and Halo Tag. POI: protein of interest. The red star represents the conjugated fluorophore. Combination of the genetical addressability of protein tags and the tunability of small molecules renders a variety of customizable methods that have greatly complemented FPs in bioimaging applications. However, the need for the addition 11 of exogenous chromophores raises the concern of cell/organelle permeability and cytotoxicity. Nonetheless, the fluorescence background remains challenging unless a fluorogenic tag is used or the unbound chromophore emits in a different spectral region from that of the fluorescent complex.85,107-109 O O N COO H S N O O O 1) Electrostatic Interaction N HS O S Cys69 O Cys69 O PYP3R N COO PYP3R H S O O 2) Increased leaving ability due to the low pKa Figure I-6. Design of the PYP-tag mutant PYP3R and its fluorogenic probe, with a focus on electrostatic interactions and the pKa value of the leaving group.110 As discussed earlier, a major attraction to the bioimaging community is the ability to turn “ON” the fluorescence signal on-demand to prevent extensive washing prior to imaging.111-115 Among all possible triggers, photochemical activation offers precise control over others. 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Methods 11, 731-U168, doi:10.1038/nmeth.2972 (2014). 28 CHAPTER II: DEVELOPING FAR-RED/NEAR-INFRARED DYE-HCRBPII FLUORESCENT TAGS FOR NO-WASH BACKGROUND-FREE LIVE CELL IMAGING APPLICATIONS Over the past 25 years, the ready availability of genetically encoded fluorescent tags has revolutionized cell biology and live-cell imaging.1-6 New tags continue to be developed that are brighter, more red-shifted, expanding the available spectral range, and better tolerated by fusions. As described in Chapter I, fluorescent proteins (FPs) provide toolkits that enable various technologies and applications in a variety of biomedical research fields.7-15 FPs are bright enough and provide emission spectra spanning the visible spectrum from the blue to the near-infrared (NIR).16-21 However, the evolving field of fluorescence microscopy demands more sophisticated FPs to address early-generation candidates' deficiencies. Optimization of characteristics such as photostability, red-shifted emission, high fluorescence quantum yield (QY), high extinction coefficient (e), and large Stokes shift (>100 nm) is always pursued. Nonetheless, certain pitfalls of FPs may hamper their application; for example, their Stokes shifts are usually small, leading to the significant overlap in the absorption and emission spectra, self- quenching, and severe limitation in the imaging depth and overall emission brightness.22-26 As discussed in Section I-2, far-red/NIR emission suffers less from interference with cellular milieu and is more advantageous for deep tissue imaging applications.27-33 Nevertheless, despite extensive efforts to develop monomeric FPs, known naturally occurring red fluorescent proteins (RFPs), such as DsRed 29 and eqFP611, tend to form obligate oligomers, which due to their large sizes, has raised serious concerns about their usefulness as molecular fusion tags in in vivo model systems.34-39 Besides, being oxygen dependent for the fluorophore maturation also limits their application in some anaerobic experiment settings.40,41 Conjugation of small synthetic dyes with genetically encodable protein/peptide tags has led to the development of flexible labeling systems that can be tailored ad infinitum and open a wide window for the development of novel imaging tags. II.1 Initial work towards developing hCRBPI-based fluorescent tags One of the main goals of our research group is to develop fluorescent tags that emit in the far-red/NIR region of the spectrum where conventional fluorescent proteins can be inadequate. Attempts towards this initiated in 2012 when our lab demonstrated the ability to regulate the absorption wavelength of all-trans-retinal in type II human cellular retinoid binding protein (hCRBPII) complexes from 425 nm to 644 nm.42 In this system, the protein was engineered to encapsulate fully and covalently bind retinal as a Schiff base (Figure II-1a). Through rational point mutagenesis, the absorption profile of the corresponding complexes shifted over a range of more than 200 nanometers as the result of altering the electrostatic environment of bound retinal within the binding pocket of the host protein (Figure II-1b). hCRBPII was selected as the target of protein engineering since it has a large binding cavity and a high tolerance for mutations that allows flexibility in the 30 protein redesign and enables binding of a wide range of different ligands.43-45 We envisioned replacing all-trans-retinal with synthetic chromophores to turn hCRBPII into a robust platform to design novel fluorescent tags. a. R58 T51 T53 T29 K40 A33 Q4 L117 Q108K Y19 Lys 108 O N H all-trans-retinal retinylidene PSB b. Figure II-1. a. Crystal structure of Q108K:K40L-hCRBPII mutant complexed with all-trans-retinal. Key residues engineered to regulate the absorption wavelength of the bound retinylidene are highlighted (PDB 4EXZ). The scheme shows the iminium (PSB) formation between lysine 108 and retinal aldehyde. b. Protein solution of different hCRBPII mutants incubated with all-trans retinal. 31 Ideally, in this approach, the unbound chromophore is not fluorescent and becomes activated only upon binding to the target protein and irradiation of the corresponding complexes. In contrast to the intrinsically fluorescent proteins that are constantly on, this method can provide spatiotemporal control of the signal. Besides, hCRBPII is a small protein (~15 KDa) that does not require oxygen to fold or bind the chromophore 46,47 and, therefore, can find potential applications in obligate anaerobes. The following briefly describes the pairing of different engineered hCRBPII mutants with various fluorophores to generate tailor-made fluorogenic protein fusion tags. As a proof of principle, in 2015, our lab successfully showed the application of merocyanine retinal analog (MCRA) as a “turn-on” fluorescent tag in conjugation with type II cellular retinoic acid binding protein (CRABPII).48 CRABPII is also a small cytosolic lipid-binding protein and is a structural homolog of hCRBPII.49,50 The spectroscopic characterizations of the complexes were examined with a variety of mutants, and their use as fluorescent tags to image E. coli was demonstrated. Nonetheless, efforts to use MCRA/CRABPII probes as a fluorescent tag to image mammalian cells were not fruitful, most probably due to protein misfolding On the contrary, hCRBPII variants showed no deleterious problem in expression or folding in mammalian cells environment. Our research group produced highly fluorescent red pigments through covalent conjugation of the merocyanine aldehyde to the active site lysine residue of hCRBPII (Figure II-2a).51 32 Upon extensive protein engineering, MCRA can bind some hCRBPII mutants almost instantaneously (less than 1 minute) with rapid cellular penetration. It binds hCRBPII as Protonated Schiff Base (PSB) under physiological pH resulting in a substantial bathochromic shift in its absorption and emission, which is entirely separated from unbound MCRA absorption and should mitigate background fluorescence from the free reagent (Figure II-2b). a. Lysine 108 CHO N hCRBPII N NH MCRA MCRA-PSB Abs λmax 603 nm Abs λmax Em λmax b. 492 nm 623 nm Figure II-2. a. Structure of MCRA and its PSB formation with active Lysine 108 of hCRBPII. b. Spectroscopic properties of free aldehyde (blue) and PSB (red). The required condition for cell imaging is 250 nM MCRA as the staining concentration for HeLa, U2OS, and COS-7 cells and 1 min incubation at 37 °C, followed by washing two times with DPBS to remove excess MCRA. However, non-specific iminium formation with other proteins in the cells leads to significant 33 fluorescent background that extra washing steps cannot diminish.51 Furthermore, the absorption maxima of the MCRA complexes with different hCRBPII variants is essentially constant (centered around 600 nm) due to the high degree of conjugation between the iminium and terminal nitrogen, and they often show Stokes shifts smaller than 30 nm. II.2 Desired features of a practical fluorescence imaging tag To this end, we showed that hCRBPII could serve as a robust platform to develop protein-based fluorescent tags; however, improving to practical tags requires particular modification of the selected fluorophore’s structure and its photophysical properties. One of the most critical issues to overcome when using tags that require supplementing exogenous synthetic ligands is the fluorescent background that can originate eighter free chromophore or off-target bindings. One way to avoid this and achieve a high signal-to-noise ratio is to use fluorescent probes that display no fluorescence until labeling occurs. Such probes are often called fluorogenic probes to highlight their ability to show an increase in fluorescence upon binding their targets.52 Fluorogenic probes can provide high sensitivity and the ability to monitor diverse events selectively and are essential components in the toolkit of chemical biology.53-55 Besides, using fluorogenic probes eliminates washing steps required for the removal of free chromophores and enables real-time measurements.56-61 Other main requirements of successful imaging tags can be summarized as followed. The probe must be bright enough to be detected well, which requires it 34 to show both high extinction coefficient and high fluorescence quantum yield as the product of these two determines the absolute brightness. High photostability that enables noninvasive long-term imaging and large Stokes shifts (typically over 80 nm) are highly preferred. As previously discussed, probes with large Stokes shifts can minimize cross-talk between the excitation source and the fluorescent emission, which generally results in poor signal-to-noise ratio and self-quenching due to back-scattering from biological samples.62 63-68 Another photophysical requirement is the far-red/NIR wavelengths. As visible light is strongly scattered in deep tissues, and thus its penetration is limited. In recent decades, the development of NIR fluorophores has enabled the visualization of deep tissues of living organisms with detailed spatiotemporal information.69-74 The tissue is more transparent to NIR (~650–900 nm) optical window light due to less absorbance by body pigments and is characterized by low autofluorescence and minimal phototoxicity for living cells.75,76 Lastly, but important, the synthetic chromophore should not be cytotoxic and possess high cell permeability. II.3 Attempts to minimize non-specific fluorescent background One of our approaches to prevent fluorescent background from non-specific iminium formation has been to design chromophores with strong solvatochromic properties. We hypothesized that using environmentally sensitive probes will enable us to tailor the specific hCRBPII/chromophore emission by changing the electrostatic environment around the chromophore inside the protein cavity. As 35 such, the fluorescence from the desired reaction is red-shifted compared to that of non-specific iminium formation. Fluorescent solvatochromic dyes are characterized as push-pull dyes containing electron-donating and electron-withdrawing groups conjugated through a π-electron spacer. These dyes indicate a feature that favors an Intramolecular Charge Transfer (ICT) process upon light excitation making these chromophores have a larger dipole moment in the excited state.77-80 Consequently, polar solvents will stabilize the excited state better than the ground state resulting in a reduction in the energy gap between the ground state and excited state energy levels, leading to bathochromic shifts in emission.81-83 Consequently, solvatochromic compounds usually show the same absorption maxima in various solvents but can have the most bathochromic shift in emission wavelength in a polar solvent environment. After an extensive search of literature for different chromophores that exhibit substantial sensitivity to solvent polarity, ThioFlour, a derivative of Dapoxyl dyes, were synthesized to pair with hCRBPII as a solvatochromic probe (Figure II-3); Dapoxyl dye family show distinctive variations in quantum yields, fluorescence wavelength maxima (i.e., larger Stokes shifts), and extinction coefficients upon changes in polarity.84-86 Conjugating the solvatochromic fluorophore ThioFluor to engineered hCRBPII mutants yielded various ThioFluor-PSB complexes with emission maxima varying from 613 nm to 744 nm, thus exhibit about 130 nm 36 variation in emission wavelength, covering both the red and far-red fluorescence wavelength regimes. N O H S N O S O R O N N Dapoxyl ThioFluor Figure II-3. Structures of Dapoxyl dye and ThioFluor. Furthermore, to prove its utility in live-cell imaging, whole-cell and subcellular compartments such as nuclei and cytosol were imaged by targeting the hCRBPII mutants with signaling peptides like nucleus-localizing sequence (NES) and nucleus-exclusion sequence (NES). However, since unbound ThioFluor is strongly emissive in hydrophobic environments, washing steps were required before fluorescence imaging experiments to remove the excess unbound dyes after incubation. EGFP-3NLS ThioFluor/hCRBPII-3NLS Figure II-4. Non-specific fluorescent background originating from off-target iminium formation in labeled HeLa cells with 10 μM ThioFluor (incubated at 37 °C for 1 hour) expressing hCRBPII-EGFP-3NLS. NLS = nuclear localization sequence. 37 Nevertheless, following optimized imaging condition, using 10 μM ThioFluor incubated at 37 °C for 1 hour provides enough time for non-specific iminium formations that emits in the same window as ThioFluor-PSB emission is collected to images cells leading in the fluorescent background even after washing steps (Figure II-4).87 However, Dr. Elizabeth Santos later successfully developed engineered hCRBPII/ThioFlour complexes as Large Stokes Shift (LSS) fluorescent tags with fluorescent properties and applications in “washed” live-cell imaging (see Chapter III). II.4 No-wash background-free fluorogenic imaging tag design An important photochemical feature of an advanced imaging tag is efficient “on/off” switching properties of the fluorescence extended from the fluorogenicity. If the fluorescence can be turned on and off iteratively by fast photochemical transformations, then the spatiotemporal control of the fluorescence signals can be realized.88 It also helps to eliminate laborious washing steps prior to imaging experiments. We have pursued developing protein-based no-wash background- free imaging via modifying the chromophore structure and reengineering the protein cavity accordingly. In this study, we have designed a chromophore that becomes fluorescent only upon binding hCRBPII mutants. It was envisioned that replacing the N,N-dimethyl amino group in ThioFluor with a phenol moiety, ThioPhenol, would make it fluorogenic (Figure II-5). Since the hydroxyl group is a weak electron donor, it fails to produce a strong ICT system, leading to a weakly emissive molecule. Furthermore, the phenol moiety's high pKa 38 prevents its deprotonation in the ground state at neutral pH. Therefore, the free aldehydic ThioPhenol does not fluoresce either in aqueous or organic solvents (<1% QY in solution). However, we envisaged that we could create a hydrogen- bonding network around the hydroxyl group through rational protein engineering and thus facilitate its deprotonation through the Excited State Proton Transfer (ESPT) process. Deprotonation of phenol moiety either through adjacent residues or water molecules upon photoexcitation liberates the negatively charged oxygen atom. The corresponding alkoxide has the same Hammett value as N,N-dimethyl amino group, i.e., both have equal electron-donating strenghths.89 H N S O S HO HO ThioPhenol ThioPhenol-SB H H N N S S HO O ThioPhenol-PSB ThioPhenolate-PSB Figure II-5. Structures of free aldehyde ThioPhenol, ThioPhenol-imine, ThioPhenol-iminium, and ThioPhenolate-iminium. On the other hand, engineering the target protein to maintain a high iminium pKa will result in Protonated Schiff Base (PSB) formation. Therefore, the resulting photoexcited product, ThioPhenolate-PSB, complex possesses a potent electron donor and electron acceptor character on both ends of the complex, forming the D−p−A structure with increased ICT emitting in far-red to NIR region of the 39 spectrum depending on the protein mutant (Figure II-5). The designed system will put the target protein in control of fluorescence selectivity and effectively diminish fluorescence from non-specific iminium formation. The same as the free aldehyde, the ThioPhenol-iminium complex (Figure II-5) is also unable to make a robust ICT and is extremely weakly fluorescent (less than 2% QY depending on the protein mutation). Thus, the designed probe is a light-triggered OFF-to-ON system that enables spatiotemporal control of the ThioPhenol/protein complex's NIR fluorescence emission as an imaging probe. II.5 Spectroscopic properties of ThioPhenol in solution First, ThioPhenol’s absorption and emission were measured in various organic solvents with different polarities to investigate its spectroscopic behavior. Results are summarized in Table II-1. Table II-1. Spectroscopic characterization of ThioPhenol in various solvents. ε Solvent λabs (nm) λem (nm) SS (nm) Φa (M-1cm-1) Toluene 386 461 75 29,715 0.00 Tetrahydrofuran 382 463 81 31,299 0.00 Ethyl acetate 378 465 87 34,319 0.00 Dimethyl sulfoxide 392 495 103 28,560 0.015 Ethanol 391 515 124 29,752 0.01 PBS buffer 389 - - 4,193 0.00 a Absolute quantum yield was measured on a Quantaurus-QY. ThioPhenol shows almost the same absorbance in different solvents, with absorption maximum centered around 390 nm (Figure II-6a). It is not soluble in 40 aqueous solutions and forms aggregates leading to its small extinction coefficient and quenched emission in PBS buffer. However, its extinction coefficient is as high as ThioFlour’s in organic solvents, about 30,000 M-1cm-1. ThioPhenol’s emission also barely changes as a function of the solvent’s ET (30) value.90 The emission maximums span a small range of 54 nm (from 461 nm in Toluene to 515 nm in EtOH) (Figure II-6b), and it hardly shows solvatochromic properties. Such behavior is expected as the hydroxyl group is a weak electron-donating group and fails to produce a strong Internal Charge Transfer (ICT) system upon excitation. Subsequently, ThioPhenol is less prone to solvent relaxations, and its emission slightly red-shifts in polar solvents. a. b. Figure II-6. Spectroscopic properties of ThioPhenol in different solvents. a. UV- Vis and b. Fluorescence spectra of ThioPhenol. In order to mimic the product of lysine 108 and free chromophore condensation in the protein pocket, the aldehyde was coupled with n-butyl amine in ethanol (Figure II-7a). The absorption and emission maxima of the 41 corresponding n-butyl imine Schiff base blue-shifts by about 25 and 50 nm, respectively. Acidification of ThioPhenol-SB in ethanol with concentrated hydrochloric acid (aq.) solution gives the protonated iminium (ThioPhenol-PSB), resulting in a large red-shift in UV-Vis with the absorption maximum at 450 nm. Furthermore, basification of the imine solution with 1M NaOH (pH>9.5-10) yields the corresponding alkoxide (ThioPhenolate-SB), showing bathochromic shifts in both absorption and emission spectra (Figure II-7b and Table II-2). a. N Bu Base S O SB-Phenolate ThioPhenolate-SB O N Bu n-BuNH2 H S S HO HO N Bu Free Aldehyde SB-Phenol S Free Aldehyde ThioPhenol-SB Acid HO PSB-Phenol ThioPhenol-PSB b. Figure II-7. a. Schiff base and protonated Schiff base (PSB) of ThioPhenol with n-butyl amine in ethanol. b. Normalized spectra of ThioPhenol and derivatives in ethanol. Absorbance (left) and emission (right). 42 Notably, the maximum emission wavelength is almost the same (~570 nm) for both ThioPhenol-PSB and ThioPhenolate-SB; however, the latter has a broad emission peak that reaches the far-red/NIR with a small shoulder around 670 nm. Nevertheless, it is not impossible to produce the ThioPhenolate-PSB complex in solution, due to lower iminium pKa (pKa~7), it will deprotonate before the phenol moiety (pKa~10) in aqueous media. As the result of failure in producing a strong ICT system, all species formed in solution and the free aldehyde are very low emissive molecules showing less than 3% quantum yield in different solvents Table II-2. This leads to essentially non-fluorescent unbound chromophore and insignificant fluorescence from off-target imine or minimum formation. Therefore, engineering hCRBPII mutants to fluoresce upon binding ThioPhenol will result in fluorogenic probes with applications in no-wash live-cell imaging. Table II-2. Spectroscopic characterization of ThioPhenol and derivatives. ε Compound λabs (nm) λem (nm) SS (nm) Φa (M-1cm-1) Free Aldehyde 391 515 124 29,752 0.01 ThioPhenol-SB 368 468 100 33,623 0.0 ThioPhenol-PSB 450 571 121 45,752 0.02 ThioPhenolate-SB 413 568 155 36,341 0.02 a Absolute quantum yield was measured on a Quantaurus-QY. II.6 General protein host properties for this study The formation of the chromophore-protein complex provides a platform to control the embedded chromophore’s photophysical properties exquisitely. The 43 protein host should fulfill certain requirements to be chosen as a good candidate for its fluorogenic partner, ThioPhenol, in this system. Fortunately, hCRBPII has a large binding cavity and a high tolerance to mutations without affecting its structure making it a robust platform for the flexible design of fluorescent tags.42,51,91 In this study, the pKa of the chromophore Schiff Base (SB) should be optimized so that an iminium is formed in neutral pH. Second, the protein cavity surrounding the hydroxyl group should provide a path for deprotonation through the ESPT process. It is essential that forming these species in the ground state will make a constantly “ON” system without any temporal control. Therefore, protein engineering should be adjusted to not deprotonate the hydroxyl group in the ground state. What follows is our approach to engineer (hCRBPII) as a host having such properties. II.7 Primary observation of dual fluorescence in hCRBPII/ThioPhenol To deprotonate the hydroxyl group in the excited state, basic or proton acceptor amino acids should be placed at its close proximity. Picking the suitable amino acids as well as their distances to the hydroxyl group is critical for success. The crystal structures of several hCRBPII mutants with ThioFluor helped to identify amino acids putatively close to the hydroxyl group. Arg 58 is located in the hCRBPII binding pocket entrance and partially covers the mouth of the cavity. Previously we reported on how its mutation to larger, hydrophobic residues could cap and sequester the binding pocket from the 44 bulk medium.42 Docking simulation of ThioPhenol in the crystal structure of Q108K:K40L:T51V:T53S:R58W-hCRBPII/ThioFluor showed that Arg 58 is at a decent distance from the hydroxyl group and can be a proper candidate to tackle (Figure II-8). R58W 3.9 Å Q108K Figure II-8. Flexible docking of ThioPhenol in the crystal structure of Q108K:K40L:T51V:T53S:R58W-hCRBPII/ThioFluor. The distance between the hydroxyl group and the indole nitrogen of 58W side chain is 3.9 Å. It was hypothesized that substitution of Arg 58 for amino acids involved in ESPT of FP’s chromophore, such as serine and glutamic acid,92 along with amino acids capable of forming a hydrogen bond network with the hydroxyl group including histidine, tryptophan, and tyrosine, could result in deprotonation of the hydroxyl group. A list of all mutations on Arg 58 is listed in Table II-3, along with the corresponding complexes' photophysical properties. For all in-protein experiments in this study, 20 μM protein and 0.5 equivalent ThioPhenol are incubated at room temperature (23 ℃) at pH ~7.2. 45 As shown in Table II-3, all mutants show absorption around 525-530 nm and emission around 580-590 nm (the blue emission). However, mutation of Arg 58 to histidine and tryptophan yielded another red-shifted emission peak around 690 nm (the red emission) (Table II-3 entries 2 and 3). Table II-3. Spectroscopic change as a result of mutating R58 in Q108K:K40L:T51V:T53S template. Entry hCRBPII Mutant λabs λem λem SS ΦESPT pKa (Blue) (Red) 1 Q108K:K40L:T51V:T53S 531 585 - 54 0.11 6.1 2 Q108K:K40L:T51V:T53S:R58W 530 586 693 56/163 0.24 6.0 3 Q108K:K40L:T51V:T53S:R58H 530 590 691 60/161 0.49 6.1 4 Q108K:K40L:T51V:T53S:R58E 519 586 - 67 0.16 5.9 5 Q108K:K40L:T51V:T53S:R58L 517 590 - 73 0.11 5.9 6 Q108K:K40L:T51V:T53S:R58Q 522 591 - 69 0.13 5.9 7 Q108K:K40L:T51V:T53S:R58K 531 583 - 52 0.16 6.8 8 Q108K:K40L:T51V:T53S:R58S 532 585 - 53 0.09 6.3 9 Q108K:K40L:T51V:T53S:R58C 522 589 - 67 0.19 6.2 10 Q108K:K40L:T51V:T53S:R58Y 522 587 - 65 0.11 7.1 We postulated that the blue emission is attributed to ThioPhenol-PSB and the red emission to ThioPhenolate-PSB (Figure II-9a), since for non-polar residues such as Leu only the blue emission is observed (Table II-3, entry 5). As explained earlier, it is not possible to form the ThioPhenolate-PSB complex in solution. Therefore we tried to prove its existence by acidifying the Q108K:K40L:T51V:T53S:R58H/ThioPhenol sample. Iminium absorption and emission peak’s intensity (at 530 nm and 590 nm, respectively) increase upon 46 direct acid titration of the sample to pH=4.7 (Figure II-9b). However, the intensity of the emission peak at 690 nm decreases with the reduction of pH. H H a. N ESPT N S S HO O ThioPhenol-PSB ThioPhenolate-PSB Em λmax Abs λmax 590 nm Em λmax 530 nm 591 nm b. 1 Figure II-9. a. ThioPhenolate-PSB, the product of ESPT process, and normalized absorption/emission spectra of the iminium of Q108K:K40L:T51V:T53S:R58H/ThioPhenol complex. b. UV-Vis (left) and fluorescence (right) spectra of the same complex upon acidification of the solution. 47 We presumed that the phenol moiety becomes deprotonated upon photoexcitation and forms the ThioPhenolate-PSB complex due to hydrogen bonding interaction between the sp2 hybridized nitrogen of histidine’s side chain and the hydroxy group of the chromophore. This nitrogen atom is neutral in physiological pH (pKa~6) and capable of building a hydrogen bond bridge with the hydroxyl group's oxygen and possibly deprotonate it in the excited state. However, the histidine side chain is protonated in acidic pHs and unable to make such interaction, and thus ThioPhenolate-PSB emission decreases at pH=4.7. Q108K:K40L:T51V:T53S:R58W/ThioPhenol mutant follows the same trend over pH changes but with lower ESPT quantum yield due to the high pKa of the nitrogen atom in the indole ring. To compare different mutants' ability to develop a hydrogen-bonding network around the hydroxyl group and generate the ESPT product, we defined ΦESPT as the fraction of total fluorescence from the excited state ThioPhenolate- PSB. Briefly, the fluorescence spectrum was deconvolved to give separate traces representing the emission from the neutral and negatively charged alkoxide, respectively. The area under each trace was then integrated to give the relative percentage of total fluorescence quantum yield. The number derived from the ThioPhenolate-PSB was determined as ΦESPT. The highest ΦESPT values (majorly yielded red-shifted emission) were obtained for hCRBPII/ThioPhenol complexes that contained R58W and R58H mutations (Table II-3, entries 2 and 3). 48 As detailed in our previous studies, bound chromophore could exist as SB or PSB; however, the current designed structure can form four different complexes depending on whether the hydroxyl group is protonated or deprotonated, ThioPhenol-SB and ThioPhenolate-SB when the chromophore is bound as imine and ThioPhenol-PSB and ThioPhenolate-PSB as iminium. Except for, ThioPhenolate-PSB, the other three complexes’ photophysical properties were measured in solution (section II.5). Nonetheless, to characterize this system in the protein cavity's complex environment, the sample was basified as well (Figure II- 10). N N S pH > 10 S HO O ThioPhenol-SB ThioPhenolate-SB Phenol-SB Phenolate-SB Phenol-PSB Figure II-10. Formation of the ThioPhenolate-SB upon basification of the solution and the UV-Vis (left) and fluorescence (right) spectra of Q108K:K40L:T51V:T53S:R58H/ThioPhenol upon base titration. 49 As shown, basification of the Q108K:K40L:T51V:T53S:R58H/ThioPhenol sample from pH=7.2 to pH=8.2 results in the disappearance of PSB absorption peak (~530 nm) and an increase in the SB absorption peak intensity at 360 nm. Further basification of the solution red-shifts the SB absorption by about 50 nm, due to the hydroxyl group deprotonation and ThioPhenolate-SB complex formation absorbing at 409 nm, very close to its absorption in EtOH solution (Table II-2). An interesting observation is that the emission intensity for the neutral complex, ThioPhenol-SB, is very low but enhances about six-fold upon deprotonation of the hydroxyl group. Such results are significantly important for imaging applications as non-specific imine formation has always been the primary source of fluorescence background in our studies. However, with the newly designed structure, it is expected to have the most negligible background fluorescence from off-target imine formation as these species are hardly emissive, and second, the emission is well-separated from the ESPT derived emission. II.8 The effect of rigidifying the chromophore through Y19W and A33W mutations on the absorption wavelength and ΦESPT In our previous studies with Professor Geiger's lab, we demonstrated that aromatic residues such as A33W and Y19W provide tight packing of the chromophore leading to chromophore rigidification and restriction. Besides, the introduction of these bulky residues results in further delocalization of the charge and a red-shift in the bound chromophore wavelength by dispelling some of the ordered water molecules out of the binding cavity as previously reported for 50 retinylidene variants.42 We chose to add these mutations, Y19W and A33W, to Q108K:K40L:T51V:T53S:R58H and Q108K:K40L:T51V:T53S:R58W templates to explore their effect on ΦESPT and other photophysical properties of the corresponding mutants (Table II-4). Table II-4. Spectroscopic change upon the addition of Y19W and A33W mutations. Entry hCRBPII Mutant a λabs λem λem SS ΦESPT pKa (Blue) (Red) 1 KLVS:R58H:Y19W 533 - 698 165 96.8 5.3 2 KLVS:R58H:Y19W:A33W 530 588 694 58/164 31.3 6.0 3 KLVS:R58W:Y19W 529 591 - 62 <2 5.7 4 KLVS:R58W:Y19W:A33W 534 594 676 60/142 <2 5.1 a KLVS equals to Q108K:K40L:T51V:T53S. No significant bathochromic shift was observed in the absorption wavelength of resultant mutants. However, interestingly, the addition of Y19W had two opposite effects on the ΦESPT of the parent mutants. It increased the ΦESPT to 95% in Q108K:K40L:T51V:T53S:R58H:Y19W mutant, the highest ΦESPT acquired up to this point, but it ceased the ESPT of the hydroxyl group and decreased the ΦESPT to less than 2% in Q108K:K40L:T51V:T53S:R58W:Y19W (entries 1 and 3). The addition of both Y19W and A33W mutations reduced the ΦESPT (entries 2 and 4). Unfortunately, the fluorescence quantum efficiency of the red-shifted emission (~690 nm) generated upon ESPT of the hydroxyl group is less than 5%, and the brightness needed to be increased to be applicable for imaging purposes. 51 We pursued two approaches to address this issue by expanding the hydrogen bonding network around the hydroxyl group and increasing the iminium pKa. II.9 Exploring the effects of L117E and L117D mutations on the iminium pKa and ΦESPT As described in Section II-6, the protein host should be reengineered to maintain a high iminium pKa (>8.5) since the fluorescent complex, ThioPhenolate- PSB, is produced upon photoexcitation of the iminium. Clearly, more iminium concentration leads to more ESPT product; however, as shown in Table II-3 and Table II-4, mutants that yielded the highest ΦESPT exhibit relatively low iminium pKa (<6), resulting in the formation of imine more than twice of iminium at neutral pH according to the according to the Henderson-Hasselbalch equation (see Figure II- 10 to compare SB vs PSB ratio). Thus, we sought to investigate the effect of high iminium pKa on the ΦESPT. Previous studies in CRABPII showed that L121E was successful at enhancing the rate of iminium formation.48 Mutation of the analogous residue, L117, in hCRBPII to aspartic acid and glutamic acid residues increased the rate of iminium formation with ThioFluor as well and retained the high iminium pKa. To this end, L117E was added to the hCRBPII mutants showing the highest ΦESPT, Q108K:K40L:T51V:T53S:R58H, and Q108K:K40L:T51V:T53S:R58H:Y19W (Table II-5, entries 1 and 2). In addition, several other mutants contained R58H and R58W and showed high iminium pKa with ThioFluor, were tested with the new structure (Table II-5, entries 3-7). 52 The PSB absorption wavelength blue-shifted by about 70 nm for entries 1- 3 and 55 nm for entries 4 and 5 upon the addition of L117E mutation. An average blue-shift of 35 nm was observed for the PSB emission. Table II-5. Spectroscopic change upon addition of L117E mutation. Entry hCRBPII Mutant a λabs λem SS ΦESPT pKa (Blue) 1 KLVS:R58H:L117E 460 552 92 <2 6.7 2 KLVS:R58H:Y19W:L117E 456 557 101 5.9 10.3 3 KLVS:R58W:Y19W:L117E 468 554 86 <2 10.0 4 KLVS:R58W:Y19W:L117E:A33W 477 563 86 <2 9.4 5 KLVS:R58H:Y19W:L117E:A33W 478 559 81 3.8 7.0 6 KLVS:R58W:Y19W:L117D:Q4F 462 549 87 <2 10.0 7 KLVS:R58H:Y19W:L117D:Q4F 378 548 178 27.3 5.4 a KLVS equals to Q108K:K40L:T51V:T53S. We hypothesized it is due to aspartate acid and glutamate's ability to localize the positive charge on iminium, as we previously showed that blue-shifted absorption wavelengths occur from localization of charge. Regardless, none of the mutants showed the red-shifted emission corresponding to the ThioPhenolate- PSB complex resulting in very low ΦESPT (<5%). As shown for KLVS:R58W:Y19W:L117E:A33W/ThioPhenol complex (entry 4), excitation of the PSB maximum yields the blue-shifted emission at 563 nm (Figure II-11). 53 Interestingly, KLVS:R58H:Y19W:L117D:Q4F mutant (entry 7) shows a very low iminium pKa, and ThioPhenol-SB is the main species at neutral pH. However, excitation of the imine at 378 nm yields the PSB emission with a large Stokes shift (178 nm) as a result of the ESPT of the hydroxyl group. For unknown reasons, it appears that increased iminium pKa stopped the ESPT of the hydroxyl group. 477 nm 563 nm Figure II-11. Absorption and emission spectra of KLVS:R58W:Y19W:L117E:A33W/ThioPhenol complex upon PSB excitation. II.9.1 Basic residues around the hydroxyl group In the next step, we attempted to expand the hydrogen bonding network around the hydroxyl group to see if the nearby basic residues could activate the bound chromophore's photoacidic properties. Fortuitously, Dr. Alireza Ghanbarpour provided the crystal structure of Q108K:K40L:T51V:T53S:R58W:Y19W:L117D:Q4F/ThioPhenol that was used to 54 investigate positions near the hydroxy group inside the hCRBPII cavity for the introduction of basic residues (Figure II-12). As shown in Figure II-12, M20, T29, A33, S76, and L77 are within 8 Å distance to the hydroxyl group, which were individually mutated to basic amino acids such as lysine, arginine, and histidine. The mutations were added to the Q108K:K40L:T51V:T53S:R58W:Y19W:L117E:A33W and Q108K:K40L:T51V:T53S:R58W:Y19W:L117D:Q4F templates that showed the highest pKa to ensure most of the bound chromophore is in the protonated state. S76 L77 4.5 Å 4.0 Å 7.9 Å Q108 T29 6.1 Å 5.9 Å M20 A33 Figure II-12. Closest residues to ThioPhenol’s hydroxyl group in the crystal structure of Q108K:K40L:T51V:T53S:R58W:Y19W:L117D:Q4F/ThioPhenol complex. Unfortunately, a number of the mutants led to insoluble protein expression; expressed proteins’ spectroscopic properties are shown in Table II-6. Nonetheless, none of the mutants found success in activating ThioPhenol-PSB 55 as a photoacid, and all of them showed the PSB emission with less than 100 nm Stokes shift and ΦESPT less than 2 percent. Table II-6. Spectroscopic properties of the protein mutants designed to increase the photoacidity of the bound ThioPhenol by introducing basic residues. Entry hCRBPII Mutant a λabs λem SS ΦESPT pKa (Blue) 1 KLVS:R58W:Y19W:L117E:A33W:L77K 471 562 91 <2 9.2 2 KLVS:R58W:Y19W:L117E:A33W:S76K 473 563 90 <2 4.8 3 KLVS:R58W:Y19W:L117E:A33W:S76R 472 564 92 3.2 9.1 4 KLVS:R58W:Y19W:L117E:A33W:S76H 472 565 93 <2 9.4 5 KLVS:R58W:Y19W:L117E:A33W:T29H 470 558 88 <2 10.1 6 KLVS:R58W:Y19W:L117E:A33W:T29R 478 569 91 <2 9.9 7 KLVS:R58W:Y19W:L117E:A33H 467 549 82 3.6 9.2 8 KLVS:R58W:Y19W:L117D:Q4F:L77H 468 557 89 <2 9.8 9 KLVS:R58W:Y19W:L117D:Q4F:S76R 464 553 89 <2 8.1 10 KLVS:R58W:Y19W:L117D:Q4F:S76H 463 559 96 3.1 7.9 11 KLVS:R58W:Y19W:L117D:Q4F:A33H 465 554 89 <2 9.5 12 KLVS:R58W:Y19W:L117D:Q4F:T29K 463 552 89 5.3 8.0 13 KLVS:R58W:Y19W:L117D:Q4F:M20K 483 562 79 <2 10.5 14 KLVS:R58W:Y19W:L117D:Q4F:M20H 464 555 91 4.8 9.3 a KLVS equals to Q108K:K40L:T51V:T53S. Presumably, due to the high pKa of lysine and arginine side chains’ (about 10.5 and 12, respectively), they are protonated in neutral pH and unable to act as a proton acceptor of the hydroxyl group upon excitation. Furthermore, although R58H mutation was the most successful in deprotonating the hydroxyl group, insertion of histidine at these selected positions was not fruitful. 56 II.9.2 Acidic residues around the hydroxyl group As described in Section I-1, Large Stokes shift fluorescent proteins such as mKeima and LSS-mKates exhibit high similarity in the interactions between the chromophores and neighboring residues. In this class of fluorescent proteins, the active site residues have been optimized such that the chromophore is stabilized through hydrogen bonding with the acidic residues (Figure II-13). Carboxylate moiety in acidic residues acts as a proton acceptor as the chromophore’s pKa is decreased several units upon excitation.93,94 Thus, we next sought to investigate whether the selected residues' mutation to aspartic and glutamic acid can enhance the ESPT process and fluorescent properties of ThioPhenol-PSB. O O Ser158 O N N O N O N H Glu160 N N H O O H O O O O Asp160 LSS-mKate 1 LSS-mKate 2 Figure II-13. Hydrogen bonding network responsible for excited state proton transfer in LSS-mKate 1 and LSS-mKate 2. Several of the designed mutants led to insoluble protein expressions, as evidenced by the presence of hCRBPII in the pellet of lysed cells via gel electrophoresis. Nevertheless, no enhancement was observed in the chromophore's ESPT activity bound to the expressed ones (Table II-7). 57 Upon examining all protein mutants and their photophysical properties till now, we realized none of the variants with high iminium pKa could yield the ESPT derived red-shifted emission. Presumably, it is the presence of L117E and L117D mutations that prevent the ESPT, although the reasons are unknown at this time. Hence, we chose to first expand a well-developed water-mediated hydrogen bonding network around the hydroxyl group, enhance the ΦESPT as much as possible, and then increase the iminium pKa through other approaches except the L117E/D mutation. Table II-7. Spectroscopic properties of the protein mutants designed to increase the photoacidity of the bound ThioPhenol by introducing acidic residues. Entry hCRBPII Mutant a λabs λem SS ΦESPT pKa (Blue) 1 KLVS:R58W:Y19W:L117D:Q4F:L77D 466 554 88 <2 8.4 2 KLVS:R58W:Y19W:L117D:Q4F:S76E 460 557 97 <2 8.5 3 KLVS:R58W:Y19W:L117D:Q4F:A33E 463 552 89 <2 8.1 4 KLVS:R58W:Y19W:L117D:Q4F:T29E 467 560 93 <2 9.6 5 KLVS:R58W:Y19W:L117D:Q4F:T29D 465 556 91 <2 9.5 a KLVS equals to Q108K:K40L:T51V:T53S. II.10 Investigating the effects of proton acceptor residues at positions A33 and F16 on the ΦESPT Among the five selected closest residues to the hydroxyl group (M20, T29, A33, S76, and L77), mutation of A33 resulted in higher protein yield and showed more stability toward the pH changes of the solution required for pKa titrations. Hence, we sought to investigate whether its mutation to proton acceptor residues such as histidine, serine, glutamic acid, and tyrosine can improve the ΦESPT. As 58 discussed previously, these residues proved to facilitate deprotonation of the phenol moiety in various FPs through direct or water-mediated hydrogen bonding in the excited state. Thus, A33 was substituted with those residues in Q108K:K40L:T51V:T53S:R58H and Q108K:K40L:T51V:T53S:R58H:Y19W templates. The highest ΦESPT was obtained with Q108K:K40L:T51V:T53S:R58H:Y19W:A33H mutant (Table II-8, entry 5). Table II-8. Spectroscopic change upon mutation of A33 to histidine, serine and glutamic acid, and tyrosine. Entry hCRBPII Mutant a λabs λem λem SS ΦESPT pKa (Blue) (Red) 1 KLVS:R58H:A33H 530 586 693 56/163 54.2 6.3 2 KLVS:R58H:A33S 529 595 691 66/162 55.1 6.5 3 KLVS:R58H:A33E 523 584 694 61/171 51.4 6.1 4 KLVS:R58H:A33Y 530 593 - 63 <2 7.1 5 KLVS:R58H:Y19W:A33H 533 588 694 55/161 96.8 6.5 6 KLVS:R58H:Y19W:A33S 534 590 693 56/159 89.1 6.4 7 KLVS:R58H:Y19W:A33E 531 593 695 62/164 95.7 6.4 8 KLVS:R58H:Y19W:A33Y 533 596 693 63/160 53.6 6.8 a KLVS equals to Q108K:K40L:T51V:T53S. As was expected, removing L117E/L117D mutations recovered the red emission, although the iminium pKa reduced by about two to three units. Alternative protein designs to increase the pKa are discussed in Section II.11.2. Most of the protein variants shown in Table II-8 provide improved ΦESPT. However, the ESPT efficiency is higher when Y19W is present. Therefore, 59 Q108K:K40L:T51V:T53S:R58H:Y19W sequence is retained in most mutants tested. Noteworthy, mutation of A33 to tyrosine enhanced the minimum pKa. To our delight, the crystal structure of Q108K:K40L:T51V:T53S:R58H:Y19W:A33Y/ThioPhenol was obtained and solved by Dr. Nona Ehyaei. Meticulous examination of the crystal structure revealed that F16 residue is very close to the hydroxyl group. Its mutation to tyrosine via mutagenesis in Pymol suggested a 3.4 Å distance, which is the shortest distance compared to previously selected positions (Figure II-14). 3.4 Å Q108K F16Y Figure II-14. The crystal structure of Q108K:K40L:T51V:T53S:R58H:Y19W:A33Y/ThioPhenol complex with F16 mutated to tyrosine via mutagenesis in Pymol. 60 Therefore, we introduced F16Y into the parent template mutant: Q108K:K40L:T51V:T53S:R58H (Table II-9, entry 1). Additionally, in order to expand the hydrogen bonding network around the hydroxyl group, A33 was mutated to amino acid residues having proton acceptor side chains in combination with F16Y. Table II-9. Spectroscopic change upon F16Y addition. Entry hCRBPII Mutant a λabs λem λem SS ΦESPT pKa (Blue) (Red) 1 KLVS:R58H:F16Y 530 596 677 66/147 62.1 7.0 2 KLVS:R58H:A33H:F16Y 533 589 677 56/144 49.8 7.2 3 KLVS:R58H:Y19W:A33Y:F16Y 531 594 678 63/147 65.2 6.9 4 KLVS:R58H:Y19W:A33W:F16Y 533 590 677 57/144 47.3 7.0 5 KLVS:R58H:Y19W:A33H:F16Y 530 - 680 150 97.6 6.9 a KLVS equals to Q108K:K40L:T51V:T53S. Remarkably, with the newly designed mutants, ΦESPT increased to more than 95% (Table II-9, entry 5), meaning almost all bound chromophores as ThioPhenol-PSB converts to ThioPhenolate-PSB upon excitation. Comparing the ΦESPT of entries 3,4, and 5 where A33 is mutated to tyrosine, tryptophan, and histidine, respectively, proves that A33H works best in conjugation with F16Y and R58H mutations. The triple mutations (F16Y, A33H, and R58H) must have aligned well toward the hydroxyl group and form a water-mediated network capable of activating the phenol moiety as a photoacid. Notably, although absorption and the blue emission wavelength corresponding to the ThioPhenol-PSB complex remained the same, the red 61 emission wavelength hypsochromically shifted about 15 nm from the parent mutants upon F16Y addition. In our previous studies with hCRBPII/retinal, Dr. Wenjing Wang demonstrated that an even distribution of electrostatic potential across the polyene led to a bathochromic shift in absorption while localizing the cation on the iminium nitrogen resulted in blue-shifted wavelengths.42 95 The emission wavelength is also correlated to the degree of intramolecular charge transfer (ICT) from the electron donor to the electron acceptor.96,97 In this system, deprotonation of the hydroxyl moiety upon excitation leads to an ICT from the correspondent alkoxide to the iminium, which results in red-shifted emission and larger Stokes shifts. A 15 nm hypsochromic shift in the emission wavelength shows that the charge transfer has decreased, and the negative charge is more localized on oxygen rather than the whole chromophore toward the iminium. We hypothesized that this is due to the electrostatic interaction between the positively charged protonated 16Y and the alkoxide group, the product of the ESPT process (Figure II-15). H H N N H S hν S O O ESPT H H O H O H Tyr 16 Tyr 16 Figure II-15. Schematic representation of the negative charge localization on the oxygen atom due to the electrostatic interaction with Tyr 16 side chain. 62 We sought to explore whether tyrosine is the best choice for position 16; hence, other proton acceptor residues such as histidine, glutamic acid, tryptophan, and cysteine were introduced to the template protein mutants, and their fluorescence properties were measured (Table II-10). Table II-10. Spectroscopic changes as a result of mutating F16. Entry hCRBPII Mutant a λabs λem λem SS ΦESPT (Blue) (Red) 1 KLVS:R58H:F16E 530 610 - 80 <2 2 KLVS:R58H:A33H:F16E 531 611 - 80 <2 3 KLVS:R58H:A33H:F16W 531 583 675 51/144 40.2 4 KLVS:R58H:Y19W:A33W:F16H 531 591 678 60/147 46.2 5 KLVS:R58H:Y19W:A33W:F16E 530 595 - 65 35.3 6 KLVS:R58H:Y19W:A33Y:F16E 530 - 72 23.2 a KLVS equals to Q108K:K40L:T51V:T53S. Unfortunately, most of the designed mutants led to insoluble protein expressions. For example, none of the protein mutants containing F16C mutation were expressed, and the ΦESPT for the successfully expressed ones was less than 50%. Presumably, since F16 is located at the interior pocket, it can be envisioned that mutation of F16 to tryptophan or histidine would lead to the steric clash of F16W/F16H with R58H and may be able to flip R58H outward and thus less ESPT of the hydroxyl group. Nevertheless, one interesting new observation was made concerning the addition of F16E. The emission of the ThioPhenol-PSB complexes red-shifts about 10 nm when F16 is mutated to glutamic acid. It seems the interaction 63 between the carboxylate and the hydroxyl group leads to a partial proton transfer. As a result, the emission wavelength (~610 nm) is between the ThioPhenol-PSB and ThioPhenolate-PSB complexes’ emission. A most interesting observation was made when overlaying the crystal structures of Q108K:K40L:T51V:T53S:R58H:Y19W:A33Y/ThioPhenol (magenta) with Q108K:K40L:T51V:T53S:R58W/ThioPhenol (green), obtained and solved by Dr. Alireza Ghanbarpour, indicates that the trajectories of 58H and 58W in these two structures are vastly different (Figure II-16). The magenta histidine adopts the flipped conformation outward to avoid the steric interaction with 33Y residue. Yet, it shows a slightly higher QESPT compared to Q108K:K40L:T51V:T53S:R58H mutant (53% VS 50%). a. b. R58H 3.6 Å A33Y R58W A33 A33Y Figure II-16. a. Overlay of Q108K:K40L:T51V:T53S:R58W/ThioPhenol (magenta) and Q108K:K40L:T51V:T53S:R58H:Y19W:A33Y/ThioPhenol (green). b. The distance between the F16Y and the hydroxyl group. 64 As R58H and A33Y showed almost the same efficiency in deprotonating the hydroxyl group, we decided to probe the effect of more tyrosine residues around the hydroxyl group on the ΦESPT. To this end, R58Y, A33Y, and F16Y mutations were added to the Q108K:K40L:T51V:T53S:R58H template (Table II-11). Table II-11. Spectroscopic changes as a result of mutating R58, F16, and A33 residues to tyrosine. Entry hCRBPII Mutant a λabs λem λem ΦESPT (Blue) (Red) 1 KLVS:R58H 530 590 691 50.2 2 KLVS:R58H:F16Y 530 596 677 62.1 3 KLVS:R58H:Y19W:A33Y 533 596 693 53.3 4 KLVS:R58H:A33Y:F16Y 536 598 - 28.8 5 KLVS:R58Y:Y19W:A33Y 531 591 - <2 6 KLVS:R58Y:F16Y 535 590 - 24.6 7 KLVS:R58Y:A33Y:F16Y 545 593 - 9.3 a KLVS equals to Q108K:K40L:T51V:T53S. Unfortunately, double or triple mutations to tyrosine did not lead to an expanded hydrogen bonding network, and in fact, the resultant mutants yielded very poor ΦESPT (Table II-11). Presumably, tyrosine residues turn away from the cavity and further from the hydroxyl group to relieve the steric hindrance. Mutants with R58Y residue resulted in the lowest ΦESPT, but the insertion of a single tyrosine at positions 16 and 33 enhances the ESPT process (entries 2 and 3). Although, as shown previously, F16Y introduces a blue-shift of 20 nm to the ESPT derived emission. 65 II.11 Attempts to enhance the red-shifted emission quantum efficiency Thus far, we have discovered that R58H is the essential mutation in the ESPT process. As shown earlier, ΦESPT would decrease dramatically or was eliminated in the absence of this mutation. We also realized that R58H, A33H, and F16Y are the three key mutations required to get the maximum ESPT quantum yield. The Q108K:K40L:T51V:T53S:R58H:Y19W:A33H:F16Y/ThioPhenol complex containing these residues yielded the highest ΦESPT (>90%); however, the ESPT derived far-red emission (680-700 nm) fluorescence quantum yield is less than 5% for this mutant. Our attempts were then focused on expanding the water-mediated hydrogen bonding network around the hydroxyl group via point mutagenesis. As we presumed, suppressing the blue-shifted non-ESPT emission and increasing the ΦESPT will lead to higher fluorescence quantum efficiency. II.11.1 Through expansion of water-mediated hydrogen bonding network around the R58H residue We sought to investigate whether providing a proton transfer network around R58H would improve the ΦESPT. It was hypothesized that hydrogen bonds from ionizable amino acids to 58H directly or through water molecules would stabilize protonated histidine in the excited state and increase its basicity. In silico modeling of R58H in the crystal structure of Q108K:K40L:T51V:T53S:R58W/ThioPhenol complex helped to identify such residues: T53S, S55, and Q38, all are located within 5 Å from the 58H, with presumably close interactions (Figure II-17a). We chose to introduce amino acids 66 having proton acceptor side chains at these positions such as histidine, tyrosine, and glutamic acid, to facilitate deprotonation of the hydroxyl group upon excitation. a. S55 Q38 2.6 Å 5.0 Å R58H 3.3 Å T53S b. R58H 3.3 Å T53S 5.5 Å Figure II-17. a. In silico modeling of R58H in the crystal structure of Q108K:K40L:T51V:T53S:R58W/ThioPhenol and the distances between S55, Q38, and T53S residues to 58H. b. T53S distances to R58H residue and ThioPhenol hydroxyl group. 67 To this point, the template Q108K:K40L:T51V:T53S was retained in all protein engineering efforts in this study, as recently Dr. Elizabeth Santos showed T51V:T53S double mutation leads to a red-shift in the absorption and emission wavelength of bound ThioFlour. In addition, T51V proved to be capable of monomerizing hCRBPII variants.87 Earlier, Dr. Wenjing Wang demonstrated that T51V mutation results in bathochromic shifts in bound retinal absorption wavelength. As seen in the crystal structure of Q108K:K40L/retinal (Figure II-18), T53 makes a water-mediated hydrogen bond with T51, which is abolished when T51 is mutated to valine, leading to the red-shifted wavelength. 4.6 Å 4.6 Å T53 2.7 Å 2.8 Å T51 Figure II-18. Crystal structures of WT-hCRBPII bound with all-trans-retinol showing the internal hydrogen bonding network among the T51 and T53 side chain residues and retinol. 68 II.11.1 A. T53 mutations T53S is situated near the phenol moiety and shows a close distance to both the hydroxyl group and 58H, 5.5 and 3.3 Å, respectively (Figure II-17b). We decided to mutate T53 to Tyr, Glu, and His residues in three different templates: Q108K:K40L:T51V:T53S:R58H, Q108K:K40L:T51V:T53S:R58H:Y19W:A33H, and Q108K:K40L:T51V:T53S:R58H:Y19W:A33W in order to study the effect of T53 mutations on emission wavelength and the ΦESPT (Table II-12). Table II-12. Spectroscopic changes as a result of mutating T53 to histidine, tyrosine, and glutamic acid. λem λem Entry hCRBPII Mutant a λabs SS ΦESPT Φ (Blue) (Red) 1 KLV:T53S:R58H 530 590 691 60/161 50.2 0.01 2 KLV:T53Y:R58H 525 594 681 69/156 48.7 < 0.01 3 KLV:T53E:R58H 526 589 703 63/177 52.3 < 0.01 4 KLV:T53S:R58H:Y19W:A33H 533 588 693 55/161 96.8 < 0.01 5 KLV:T53H:R58H:Y19W:A33H 520 586 691 66/171 48.9 < 0.01 6 KLV:T53Y:R58H:Y19W:A33H 527 590 693 63/166 91.6 < 0.01 7 KLV:T53E:R58H:Y19W:A33H 531 - 704 60/173 97.8 0.01 8 KLV:T53S:R58H:Y19W:A33W 530 588 694 58/164 31.3 0.01 9 KLV:T53H:R58H:Y19W:A33W 521 592 - 71 18.7 < 0.01 10 KLV:T53Y:R58H:Y19W:A33W 528 599 690 71/162 68.3 < 0.01 11 KLV:T53E:R58H:Y19W:A33W 521 587 702 66/180 84.8 0.01 a KLV equals to Q108K:K40L:T51V. 69 As shown, T53H mutation reduced the ΦESPT dramatically (entries 5 and 9); it also decreased the protein expression yield. On the other hand, T53E mutation enhanced both ESPT and fluorescence quantum efficiency (entries 3, 7, and 11). T53Y mutation does not provide such interaction, and proteins with 53Y show almost the same ΦESPT as their parent templates. Another interesting observation is that T53E mutation bathochromicaly shifts the ESPT emission by about 7 nm and yields the most red-shifted emission wavelength (entries 3, 7, and 11). The glutamate side chain must be oriented toward the hydroxyl group facilitating its deprotonation leading to a stronger ICT. Table II-13. Spectroscopic changes as a result of mutating T53. λem λem Entry hCRBPII Mutant a λabs SS ΦESPT Φ (Blue) (Red) 1 KLVH:T53S:Y19W:A33H:F16Y 530 - 680 150 97.6 0.01 2 KLVH:T53Y:Y19W:A33H:F16Y 525 581 682 56/157 98.2 0.01 3 KLVH:T53E:Y19W:A33H:F16Y 524 583 681 59/157 97.5 0.01 4 KLVH:T53S:Y19W:A33W:F16Y 533 590 677 57/144 47.3 0.01 5 KLVH:T53E:Y19W:A33W:F16Y 521 592 679 71/158 46.9 < 0.01 6 KLVH:T53S:Y19W:A33Y 533 596 693 63/160 53.3 0.02 7 KLVH:T53E:Y19W:A33Y 526 590 702 64/176 78.2 0.01 8 KLVH:T53S:A33H:F16Y 533 589 677 56/144 49.8 0.02 9 KLVH:T53E:A33H:F16Y 525 588 682 63/157 57.1 < 0.01 10 KLVH:T53S:A33H 530 586 693 56/163 54.2 0.01 11 KLVH:T53E:A33H 525 588 702 63/177 66.9 0.01 a KLVH equals to Q108K:K40L:T51V:R58H. To further explore the effect of T53E and T53Y mutations on the formation of ThioPhenolate-PSB and the fluorescence quantum yield, other protein 70 templates were tested, and their spectral properties were compared to the parent mutants (Table II-13). As was observed before, a blue-shift of 15-20 nm happens upon the addition of F16Y mutation; for example, see entries 5 and 6, Table II-13. Nevertheless, unfortunately, neither quantum efficiency nor ΦESPT changed markedly in these newly designed mutants. Q108K:K40L:T51V:T53E:R58H:Y19W:A33H/ThioPhenol complex yielded the highest ΦESPT and fluorescence quantum efficiency when comparing to the other protein mutants in Tables II-12 and II-13. However, the addition of F16Y to this mutant did not enhance its fluorescence properties (Table II-13, entry 3). In general, it seems that in this study, the effect of each individual residue does not add up, and it is the combination of residues that improve the ESPT process. II.11.1 B. S55 mutations As was indicated in Figure II-17a, another residue in the close vicinity of R58H is S55, which shows a distance of 2.6 Å to the histidine side chain. However, unfortunately, the expansion of the hydrogen bonding network through this residue was unsuccessful. S55 mutation leads to majorly dimer formations (Table II-14), which are not suitable for imaging purposes. Nonetheless, we decided to measure the fluoresce properties of the PSB in the expressed dimers to see how the ΦESPT varies compared to the monomeric forms of the protein (Table II-15). 71 As shown, dimeric proteins showed low ΦESPT; another interesting new observation is that even for the same mutant, the dimer shows less ΦESPT than the monomeric form (Table II-15, entries 3 and 4). While the dimeric species of hCRBPII are not desirable, these results seem to indicate that ΦESPT is critically dependent on the environment around the bound chromophore, changing one residue even if it is far from the hydroxyl group can affect its deprotonation. Table II-14. Protein expression yields of hCRBPII mutants upon S55 mutation to histidine, tyrosine, and glutamic acid. Monomer Dimer Mol% hCRBPII Mutant a (mg/L) (mg/L) dimer KLVS:S55E:R58H:A33H - 15 .4 > 98 KLVS:S55Y:R58H - 19.2 > 98 KLVS:S55E:R58H 3.1 16.2 > 95 KLVS:S55H:R58H - 17.3 > 98 a KLVS equals to Q108K:K40L:T51V:T53S. Table II-15. Spectroscopic properties of the PSB as a result of mutation of S55. Entry hCRBPII Mutant a λabs λem λem SS ΦESPT (Blue) (Red) 1 KLVS:S55E:R58H:A33H (dimer) 530 590 - 60 27.4 2 KLVS:S55Y:R58H (dimer) 530 591 - 61 18.5 3 KLVS:S55E:R58H (dimer) 529 589 - 60 31.2 4 KLVS:S55E:R58H (monomer) 523 590 674 67/151 50.2 5 KLVS:S55H:R58H (dimer) 532 593 - 61 22.7 a KLVS equals to Q108K:K40L:T51V:T53S. 72 II.11.1 C. Q38 mutations A water-mediated hydrogen bonding network between Q38 and Q128 is observed in most hCRBPII mutants if the two residues are maintained (Figure II- 19). Q128 3.0 Å 3.2 Å 2.7 Å 2.9 Å Q38 Figure II-19. Water mediated hydrogen bonding between Q38 and Q128 in the crystal structure of hCRBPII-Q108K:K40L/retinal PDB 4EXZ. The addition of T53S mutation disturbs part of the conserved water- mediated network through the formation of another tight hydrogen bond between T53S and Q38. An overlay of the crystal structure of Q108K:K40L/retinal with Q108K:K40L:T51V:T53S:R58W/ThioPhenol indicates that while Q128 and Q38 maintain almost the same conformation in both structures, the 53S side chain has rotated toward Q38 to form the hydrogen bond with Q38 (Figure II-20). 73 As shown earlier in Figure II-17, the hydrogen bonding interaction between Q38 and R58H leads to the imidazoline ring of the histidine to adopt a parallel trajectory toward the phenol moiety. We sought to examine whether Q38 mutation and elimination of this interaction would result in the imidazoline ring rotation toward the hydroxyl group and consequently shorter distance. Table II-16. Spectroscopic properties of the PSB as a result of mutation of Q38. λem λem Entry hCRBPII Mutant a,b λabs SS ΦESPT Φ (Blue) (Red) 1 KLVSH:Q38H 515 588 - 73 9.8 < 0.01 2 KLVSH:A33H:Q38Y 525 591 685 66/160 48.7 < 0.01 3 KLVSH:Q38E 528 586 689 58/161 49.8 < 0.01 4 KLVSH:F16Y:Q38E 533 595 674 62/141 48.3 0.02 5 KLVSH:A33H:F16Y:Q38E 534 596 673 62/139 47.2 0.01 6 KLVSHW:A33H:F16Y:Q38E 533 - 677 144 66.4 0.01 7 KLVSHW:A33Y:F16Y:Q38E 529 588 678 59/149 51.5 0.01 a KLVSH equals to Q108K:K40L:T51V:T53S:R58H. b KLVSHW equals to Q108K:K40L:T51V:T53S:R58H:Y19W/ Mutation of Q38 to histidine in Q108K:K40L:T51V:T53S:R58H template significantly reduced ΦESPT from 50% to less than 10% (Table II-16, entry 1). Moreover, Q108K:K40L:T51V:T53S:R58H:A33H:Q38Y showed diminished ΦESPT and fluorescence Φ (Table II-16, entry 2). In contrast, Q38E/ThioPhenol showed elevated ΦESPT and, more importantly, the fluorescence Φ (Table II-16, entry 3). However, the addition of Q38E to other templates did not improve the fluorescent properties compared to the parent mutants. 74 a. T53S T53 Q38 Q128 b. T53S 2.7 Å Q38 3.2 Å 2.6 Å 2.6 Å Q128 Figure II-20. a. Overlay of Q108K:K40L:T51V:T53S:R58W/ThioPhenol (green) and Q108K:K40L/retinal (pink). b. Water network between T53S, Q38, and Q128 in Q108K:K40L:T51V:T53S:R58W/ThioPhenol crystal structure. 75 Nonetheless, an interesting trend was observed in the absorption and emission of mutants containing Q38E upon acidification. During acidifying these complexes to pH less than 6, a small shoulder appears to the right side of the PSB absorption peak. Notably, the excitation of this shoulder around 610 nm leads to emission maximized at the same wavelength when PSB is excited. However, with lower intensity (Figure II-21b), thus, we surmised the shoulder corresponds to ThioPhenolate-PSB produced in the ground state (see Section II.11.3 C for more data and discussion). a. b. Figure II-21. a. UV-Vis (left) and fluorescence (right) spectra of Q108K:K40L:T51V:T53S:R58H:A33H:F16Y:Q38E/ThioPhenol upon acid titration. b. Excitation of the shoulder indicated by the black arrow results in the same emission as PSB excitation. 76 Furthermore, as opposed to previous observations, protein variants containing Q38E showed a remarkable increase in the red-shifted emission upon acidification. For example, in Q108K:K40L:T51V:T53S:R58H:A33H:F16Y:Q38E mutant, the ΦESPT increases from 45% in pH 7.3 to more than 90% in pH 5.3 (Figure II-21a). As discussed earlier in this section, we hypothesize that the lower pKa of the Glu side chain next to 58H produces a hydrogen bond network and facilitates the proton transfer from the hydroxyl group in acidic pH. Unfortunately, there are no crystal structures containing R58H and Q38E mutations with ThioFluor or ThioPhenol to prove this claim. Among all protein mutants designed to extend the water-mediated hydrogen bonding network around R58H and the hydroxyl group, only the ones that contained Q38E and T53E mutations showed enhanced properties. Hence, we decided to measure the fluorescence characteristics of variants that contained both mutations together (Table II-17). Table II-17. Spectroscopic change as a result of mutation Q38 and T53 mutation to Glu. Entry hCRBPII Mutant a λabs λem λem ΦESPT Φ (Blue) (Red) 1 KLV:T53E:R58H:Q38E 527 588 709 49.1 < 0.01 2 KLV:T53E:R58H:Y19W:A33H:Q38E 526 - 707 97.8 0.01 a KLV equals to Q108K:K40L:T51V. The combination of the two mutations resulted in the most red-shifted emission wavelengths (>705 nm); however, only with the presence of A33H fluorescence properties improved compared to the parent mutant. Therefore, it 77 cannot be concluded that the addition of Q38E and T53E effects are additive and would always result in brighter complexes. II.11.2 Enhancing the ESPT emission quantum efficiency through increasing the iminium pKa To this end, we could design several ThioPhenol/hCRBPII complexes showing more than 99% ΦESPT through rational point mutagenesis. Yet, it was necessary to increase the fluorescence quantum yield. As discussed previously, increasing the iminium pKa is another approach for enhancing the QY. Unfortunately, for the protein mutants that yielded the highest ΦESPT, the iminium pKa was determined to be in the range of 5.0 to 6.5, which leads to the SB concentration more than twice of the PSB’s according to the Henderson- Hasselbalch equation. An interesting observation was made when comparing the crystal structures of ThioPhenol and ThioFluor bound to Q108K:K40L:T51V:T53S:R58W:Y19W:L117D:Q4F mutant. Both chromophores and the 117D residue adopt the same conformation, but the iminium’s configuration is different. ThioPhenol forms a cis iminium, while ThioFluor adopts the trans isomer (Figure II-22a). As shown, there is a closer distance between L117D and the iminium nitrogen atom in the trans isomer, 4.5 Å compared to 5.3 Å, which presumably should lead to the decreased pKa. Our lab also has formerly observed the trend that for both CRABPII and hCRBPII bound to retinal, cis iminiums show a higher pKa than the trans iminiums; 98,99 a similar trend is 78 observed with ThioFluor as well. Nonetheless, the pKa of the cis isomer formed upon binding to ThioPhenol is lower by 1 unit. Generally, it was observed that ThioPhenol tends to show lower iminium pKa compared to ThioFluor when bound to the same mutant. a. Q108K L117D b. 4.5 Å 5.3 Å L117D L117D Figure II-22. a. An overlay of crystal structures of Q108K:K40L:T51V:T53S:R58W:Y19W:L117D:Q4F/ThioPhenol (purple) and ThioFluor (green) showing cis and trans iminium conformation, respectively. b. The distance between L117D residue and the iminium nitrogen atom for ThioFluor complex (left) and ThioPhenol (right). As shown in Section II-9, the introduction of L117E or L117D mutations accelerate the iminium formation process and provides high iminium pKa values. 79 However, in this system, the addition of L1117E/D mutations prevent from deprotonation of the hydroxyl group upon excitation (see Tables II-5 and II-6). Hence, we sought to investigate whether mutation of other residues in the vicinity of the iminium to acidic amino acids can provide a high pKa without impeding the QESPT. As seen in the crystal structure of Q108K:K40L:T51V:T53S:R58H:Y19W:A33Y/Thiophenol, Q4, I42, 51V, F64, L93, L115, and L117 are all within 6 Å from the iminium (Figure II-23). F64 T51V L93 I42 Q4 L117 L115 Figure II- 23. Highlighted residues mutated to glutamic acid in order to interact with the iminium to increase its pKa. All these residues are located in the hydrophobic protein binding pocket, and it is widely accepted that the hydrophobic interactions can influence protein folding and stability. 100-102 It was more of a challenge to express soluble proteins 80 when substituting non-charged hydrophobic residues with polar acidic residues. Unfortunately, except for L117E mutation, most of the designed mutants led to insoluble protein expression (Table II-18), misfolded and aggregated in the form of insoluble inclusion bodies during protein expression, and so exploration at these positions could not be followed. Table II-18. List of mutants that resulted in insoluble protein expression upon introduction of Glu resides in the proximity of iminium. Entry hCRBPII Mutant a 1 KLVS:R58H:Y19W:A33W:F16Y:Q4E 2 KLVS:R58H:I42E 3 KL:T51E:T53S:R58H 4 KLVS:R58H:A33W:Y19W:F16Y:F64E 5 KLVS:R58H:L93E 6 KLVS:R58H:A33W:Y19W:F16Y:L115E a KL equals to Q108K:K40L and KLVS equals to Q108K:K40L:T51V:T53S. II.11.2 A. L117 mutation We then sought to explore if other residues at Leu 117 can stabilize the PSB. As is shown in Figure II-22b, the distance between L117D residue and the iminium nitrogen is not that close to interacts directly, 5.3 Å, and thus it is presumably a water-mediated stabilization. It was envisioned that the introduction of other ionizable amino acids such as Ser, Thr, Gln, Try, His, and Cys residues at position 117 might form such water network and increase the iminium pKa without 81 the prevention of the ESPT process. The spectroscopic properties of all expressed mutants are listed in Table II-19. Table II-19. Spectroscopic properties of protein mutants upon mutation of L117 in order to increase the iminium pKa. λem λem Entry hCRBPII Mutant a,b λabs ΦESPT Φ pKa (Blue) (Red) 1 KLVSH:Q38E 528 586 689 49.8 < 0.01 5.9 2 KLVSH:Q38E:L117T 487 564 - 16.4 0.09 7.6 3 KLVSH:Q38E:L117H 483 576 - 28.2 0.11 5.6 4 KLVSH:Q38E:L117C 513 580 - 31.5 0.07 8.1 5 KLVSH:F16Y 530 596 677 62.1 0.04 7.1 6 KLVSH:F16Y:L117Y 532 598 - 8.4 0.02 8.2 7 KLVSH:F16Y:L117C 480 581 679 34.2 0.07 8.6 8 KLVSH:A33H:F16Y 533 589 677 49.8 0.02 7.3 9 KLVSH:A33H:F16Y:L117T 505 573 - 25.3 0.04 7.8 10 KLVSH:A33H:F16Y:L117Y 536 595 - 4.3 0.09 8.6 < 11 KLVSH:A33H:F16Y:L117S 515 562 - 0.05 6.9 0.02 12 KLVSH:A33H:F16Y:L117C 523 579 678 54.6 0.05 8.8 13 KLVSH:Y19W:A33H:F16Y 530 - 680 97.6 0.01 7.0 14 KLVSH:Y19W:A33H:F16Y:L117T 498 568 - 19.8 0.08 7.7 15 KLVSH:Y19W:A33H:F16Y:L117S 492 571 - 8.6 0.07 8.1 16 KLVSH:Y19W:A33H:F16Y:L117E 511 564 - 5.2 0.06 7.5 17 KLVSH:Y19W:A33H:F16Y:L117C 520 577 680 95.5 0.08 8.6 18 KLV:T53E:R58H:Y19W:A33H 531 - 704 97.8 0.01 6.0 19 KLV:T53E:R58H:Y19W:A33H:L117Q 470 560 670 37.6 0.02 5.8 20 KLV:T53E:R58H:Y19W:A33H:L117H 361 473 - - 0.03 5.3 21 KLV:T53E:R58H:Y19W:A33H:L117C 505 573 698 48.3 0.02 5.5 a KLV equals to Q108K:K40L:T51V. KLVSH equals to Q108K:K40L:T51V:T53S:R58H. 82 We chose to make L117 mutations with five of the protein templates that showed higher ΦESPT. Substitution of hydrophobic leucine at 117 with ionizable residues such as Thr, His, and Cys led to a blue-shift in the absorption wavelength of 41 nm, 45 nm, and 15 nm from the parent mutant Q108K:K40L:T51V:T53S:R58H:Q38E, respectively (Table II-19, entries 2, 3 and 4). Presumably, the introduction of polarizable amino acids and the carboxylate from Q38E in the proximity of the iminium localizes the positive charge, leading to the observed blue shift. Proposedly, for the same reasons, L117 mutations to Gln and Cys on the Q108K:K40L:T51V:T53E:R58H:Y19W:A33H:L117C template containing T53E resulted in hypsochromic shifts in the absorption wavelength, 61 nm, and 26 nm respectively (entries 19 and 21). Although Q38E and T53E mutations initially increased the ΦESPT, it was observed that the polarity changes in the protein cavity, as a result of the presence of their carboxylate side chain plus polar residues at position 117 exerts a significant impact on the absorption and emission profile of ThioPhenol-PSB and its ΦESPT. The introduction of L117T did not change the iminium pKa, but it reduced the ΦESPT dramatically compared to the parent mutants (entries 2, 9, and 14). Interestingly, the intensity of the blue emission corresponding to the ThioPhenol- PSB complex increased drastically upon L117T addition, while the red-shifted ESPT emission intensity remained the same, leading to decreased ΦESPT (Figure II-24). Unfortunately, the addition of L117H and L117Q severely hampered the chromophore’s binding; monitoring the SB/PSB formation via the UV-Vis 83 absorption peak showed that even after 12 hours of incubation, part of the chromophore remained unbound. Nonetheless, the iminium pKa of the produced complexes was determined to be lower than the initial mutants; for instance, binding to Q108K:K40L:T51V:T53E:R58H:Y19W:A33H:L117H mutant resulted in mere SB formation (entry 20). Figure II-24. UV-Vis (left) and fluorescence (right) spectra of Q108K:K40L:T51V:T53S:R58H:Y19W:A33H:F16Y/ThioPhenol (red) and Q108K:K40L:T51V:T53S:R58H:Y19W:A33H:F16Y:L117T/ThioPhenol (blue). Inclusion of L117Y and L117S mutations led to relatively high iminium pKa values; for example, a pKa of 8.5 was observed with Q108K:K40L:T51V:T53S:R58H:F16Y:L117Y, which is higher by about two units as compared to its parent mutant (entry 10). Regardless, both Tyr and Ser at position 117 suppressed the ESPT emission and led to ΦESPT < 0.05 (entries 6, 10, 11, and 15). Fortuitously, however, mutation of Leu 117 to cysteine yields the best results; the iminium pKa increased to more than 8, leading to the PSB as the major component. As a result, the emission intensity enhanced significantly; it led to 84 almost 2, 4, and 5-fold emission enhancement for entries 7, 12, and 17, respectively. To our delight, while the introduction of L117C provides mutants with higher fluorescence quantum yield, it does not affect the ΦESPT efficiency. Excellent data was acquired with Q108K:K40L:T51V:T53S:R58H:Y19W:A33H:F16Y:L117C mutant, the iminium pKa increased to 8.6 with the ΦESPT as efficient as 95%, and the fluorescence quantum yield elevated to 8% (entry 17). Interestingly, the substitution of glutamic acid with cysteine in the same mutant shows a high pKa of 7.5; however, as previously reported, the ESPT emission was absent (entry 16). As only the addition of L117C to protein variants contained the Q108K:K40L:T51V:T53S:R58H pentamutant, resulted in high iminium pKa and enhanced fluorescence properties, this template was retained in most of the further protein engineering. Great results with L117C piqued our interest to explore the effects of Leu 117 mutation to methionine on the iminium pKa and the ΦESPT. The resultant mutant Q108K:K40L:T51V:T53S:R58H:Y19W:A33H:F16Y:L117M showed slightly higher iminium pKa but lowered fluorescence quantum yield Table II-20. Table II-20. Spectroscopic properties as the result of L117M mutation. Entry hCRBPII Mutant a λabs λem λem ΦESPT Φ pKa (Blue) (Red) 1 KLVSHWH:F16Y:L117C 520 577 680 95.5 0.08 8.6 2 KLVSHWH:F16Y:L117M 525 583 672 84.3 0.05 8.9 aKLVSHWH equals to Q108K:K40L:T51V:T53S:R58H:Y19W:A33H. 85 Fortunately, Dr. Nona Ehyaei was able to obtain the crystal structure of Q108K:K40L:T51V:T53S:R58H:Y19W:A33H:F16Y:L117C/ThioPhenol, in which the 117C shows a distance of 5.4 Å to the iminium nitrogen atom (Figure II-25a). In addition, as shown, the cis iminium is stabilized through a water-mediated hydrogen bond with the Q4 residue. Previously, Dr. Wenjing Wang and Dr. Elizabeth Santos noticed the same interaction is stabilizing the retinal/ThioFluor’s PSB bound to hCRBPII. Elimination of the water-mediated interaction through mutation of Q4 to Phe results in the chromophore movement toward the mouth of the protein cavity, which results in more exposure to the bulk solvent and non-radiative relaxation pathways. In addition, An overlay of the crystal structures of Q108K:K40L:T51V:T53S:R58H:Y19W:A33H:F16Y:L117C/ThioPhenol with Q108K:K40L:T51V:T53S:R58W:Y19W:L117D:Q4F/ThioPhenol shows that the distance between 117C/117D and the corresponding iminium nitrogen is almost the same. However, mutation of F16 to Tyr in Pymol for the latter complex shows a further distance to the hydroxyl group, 3.9 Å vs. 4.5 Å, respectively (Figure II- 25b). These observations help to understand that the improved hydrogen bonding network around the hydroxyl group and stabilizing the iminium positive charge led to the increased ΦESPT and the fluorescence efficiency with the former complex. 86 a. 2.8 Å 2.9 Å 5.4 Å Q4 L117C b. 4.5 Å Q4 3.9 Å 5.3 Å L117C 5.4 Å L117E F16Y Figure II-25. a. Water mediated hydrogen bonding between Q4 and the iminium, and the distance between L117C and the iminium nitrogen atom in the crystal structure of Q108K:K40L:T51V:T53S:R58H:Y19W:A33H:F16Y:L117C/ThioPhenol complex. b. The different trajectory of chromophore shown upon the overlay of Q108K:K40L:T51V:T53S:R58H:Y19W:A33H:F16Y:L117C/ThioPhenol (green) and Q108K:K40L:T51V:T53S:R58W:Y19W:L117D: Q4F/ThioPhenol (purple) with F16 residue mutated to Try in the latter (shown in grey). 87 Notably, although ThioFluor forms a trans iminium in the presence of L117D/E mutations (Figure II-22a); analysis of all crystal the structures acquired from hCRBPII/ThioPhenol complexes shows a cis minimum with all mutants. II.11.2 B. Q4 and T51 mutation Next, we sought to explore whether mutation of Gln 4 to ionizable amino acids can strengthen the water-mediated interaction with PSB and increase the iminium pKa. Most of the designed mutants led to insoluble protein expression or proteins that precipitated during binding with ThioPhenol. However, all expressed mutants showed low iminium pKa (~5), with no PSB apparent at neutral pH. The photophysical properties of the SBs were measured and are listed in Table II-21. Table II-21. Spectroscopic properties of the SB as the result of Q4 mutation. Entry hCRBPII Mutanta λabs λem pKa 1 KLVSH:Y19W:A33H:F16Y:Q4T 360 477 4.9 2 KLVSH:A33H:F16Y:Q4S 365 467/528 5.3 4 KLV:T53E:R58H:Y19W:A33H:Q4Y 361 458 - aKLV equals to Q108K:K40L:T51V, and KLVSH equals to Q108K:K40L:T51V:T53S:R58H. In Section II-11, we discussed the effects of T51V mutation on the absorption and emission wavelength of the corresponding complexes and its ability in monomerizing hCRBPII variants. As shown in Figure II-23, T51V is one of the closest residues to the iminium nitrogen atom, 4.7 Å, and prone to impact the pKa. Previously Dr. Elizabeth Santos illustrated that acidic residues at position T51 close to the putative aldehyde binding site facilitate the PSB formation 88 considerably, presumably through activation of the retinal aldehyde group. Additionally, the introduction of polar residues in close proximity to the iminium localizes the charge, leading to blue shift in wavelength. Nonetheless, we decided to explore the effect of introducing the acidic and polarizable amino acids such as cysteine and methionine on the iminium pKa in this system. The mutation was done on Q108K:K40L:T51V:T53S:R58H:F16Y:Y19W:A33H:L117C template that showed the highest ΦESPT (Table II-22). Both the iminium pKa and ΦESPT were enhanced upon introduction of the T51M mutation (entry 2). Mutation of T51 to methionine via mutagenesis in Pymol suggests almost the same distance as T51V (4.4Å); however, the polarizability of the sulfur atom in the methionine side chain can stabilize the iminium and explain the higher iminium pKa. Table II-22. Spectroscopic change as the result of the introduction of T51M mutation. Entry hCRBPII Mutant λabs λem λem ΦESPT pKa (Blue) (Red) 1 T51V 520 577 680 95.5 8.6 2 T51M 523 - 674 >99 9.0 II.11.2 C. K40 Mutation As our last attempt to increase the pKa, we chose to manipulate the environment around the iminium through K40 mutation. K40L has been retained in all protein mutants studied in this chapter. In studies with hCRBPII/retinal, Dr. Wenjing Wang previously demonstrated that K40L increases the pKa of retinal- PSB, 8.5 for Q108K:K40L compared to <6.5 for Q108K. Additionally, it was shown 89 that Lys 40 was disturbing the stability of the apo protein upon introduction of Lys108, due to charge repulsion of the two residues in close proximity. Q108K 4.6 Å K40E Figure II-26. The distance between the K40L residue and the iminium nitrogen atom in the crystal structure of Q108K:K40L:T51V:T53S:R58H:Y19W:A33H:F16Y:L117C/ThioPhenol. K40 residue shows a close distance of 4.6 Å with the iminium nitrogen (Figure II-26), and as discussed above, mutation of K40 to leucine increased the retinal-PSB pKa remarkably. Therefore, we sought to investigate whether ionizable residues at position 40 are capable of stabilizing the iminium without reducing the ΦESPT. For the sake of comparison, K40 mutations were done on the protein template yielded the best results, Q108K:K40L:T51V:T53S:R58H:Y19W:A33H:F16Y:L117C. Several neutral polar residues were tested since the positive charge on the basic residues’ side chain can destabilize the PSB, as discussed above. As the protein yield was low for some of the mutations, the Q4F mutation was included since it has been shown to lead to higher protein expression yields without 90 negatively affecting the complex’s quantum yield (denoted by a star in Table II- 23). The photophysical and iminium pKa of the expressed mutants are listed in (Table II-23). Table II-23. Spectroscopic properties as the result of K40 mutation. Dipole Entry Residue at 40a λabs λem ΦESPT pKa moment (D) 1 K40L 520 577 95.5 8.6 3.78 2 K40T 495 572 22.6 6.7 9.30 3 K40S* 493 574 28.6 5.9 9.84 4 K40Y 505 575 10.5 7.9 10.41 5 K40C* 500 570 9.3 6.4 10.74 6 K40N* 370 465 < 0.02 5.0 18.89 7 K40D 460 550 4.3 5.3 29.49 8 K40Q 480 564 < 0.02 >5 39.89 9 K40H* 490 576 19.4 5.7 20.44 a The template for starred mutants is Q108K:K40L:T51V:T53S:R58H:F16Y:Y19W:A33H:L117C:Q4F, and for others is Q108K:K40L:T51V:T53S:R58H:F16Y:Y19W:A33H:L117C. All mutations showed hypsochromic shifts in the absorption wavelength relative to the K40L template due to the localization of the positive charge on the iminium nitrogen atom. For instance, K40D mutation resulted in the most blue- shifted PSB absorptions wavelength; negative charge of the aspartic acid side chain exerts an electrostatic interaction on the PSB that leads to iminium stabilization and the observed blueshift (entry 7). Protein variant with K40Q mutation had the lowest iminium pKa resulting in mere SB formation (entry 8). Interestingly it seems there is a correlation between the residue’s dipole moment 91 value and the complex absorption wavelength; in general, less polar residues at position 40 show more red-shifted absorption wavelengths due to the same results discussed above. Nonetheless, the ΦESPT of the designed mutants was diminished drastically (<0.30) compared to the parent mutant. There have been many attempts to analyze and optimize ThioPhenol’s iminium pKa following several different approaches. Our studies show that the conventional mutations proved to increase the pKa for retinal and ThioFluor such as L117D/E have a detrimental effect on the ΦESPT of the current system. The best results were obtained upon the combination of Q4, K40L and L117C mutations, which give rise to the maximum pKa of 9.6. II.11.3 Through expanding water-mediated network around the hydroxyl group In the early stages of this study, several spots (M20, T29, A33, S76, and L77) were recognized as the candidates for point mutagenesis to facilitate the deprotonation of the hydroxyl group upon excitation (Figure II-12). However, later we realized most of those mutations were done on protein templates not optimized to yield high ΦESPT due to the presence of L117D or L117E. Unfortunately, mutation of M20, S76 primarily resulted in very low protein expressions. Hence, we chose to mutate T29 and L77 to proton acceptor residues in the newly designed templates. 92 II.11.3 A. L77 mutation In Section II-9-1, it was shown that the L77 residue is close to the hydroxyl group (4Å, see Figure II-12); however, attempts to enhance the ΦESPT through its mutation to basic residues were not fruitful, as later we realized L117E or L117D mutations inhibit the process of proton transfer from the hydroxyl group to its neighboring residues upon excitation. Now that we could successfully design protein mutants showing more than 99% ΦESPT through rational point mutagenesis, we south to investigate whether mutation of L77 to proton acceptor residue will improve the fluoresce quantum efficiency of the corresponding complexes. None of the protein variants contained L77C, and L77M led to soluble protein expressions; L77H mutation also resulted in low protein yield. Photophysical properties of the expressed mutants are listed in (Table II-24). Table II-24. Spectroscopic change as the result of L77 mutations. Entry hCRBPII Mutant a λabs λem λem ΦESPT Φ (Blue) (Red) 1 KLVS:R58H:A33H 530 586 693 54.2 0.01 2 KLVS:R58H:A33H:L77H 536 584 693 53.2 0.02 3 KLVS:R58H:A33H:L77Y 520 584 692 64.8 0.05 4 KLVS:R58H:F16Y:L77Y 512 587 673 88.7 0.02 a KLVS equals to Q108K:K40L:T51V:T53S, 3 The addition of L77H mutation resulted in almost the same properties (compare entry 1 with entry 2). On the other hand, mutation to tyrosine enhanced both the ΦESPT and fluorescence quantum efficiency. L77Y, in combination with F16Y, improved the ΦESPT to more than 85%, although the inclusion of F16Y led to 93 the 20 nm blue-shift in the ESPT emission wavelength (entry 4). The highest Φ was observed for Q108K:K40L:T51V:T53S:R58H:A33H:L77Y mutant (entry 3). Comparing the properties of entry 3 and 4 shows that inclusion of A33H improves the fluorescence efficiency, while F16Y enhances the ΦESPT. Therefore, we sought to investigate the effect of L77Y when both mutations are present on the template showed the highest pKa Q108K:K40L:T51V:T53S:R58H:Y19W:A33H:F16Y:L117C (Table II-25). The resultant mutant was less stable, evident from the absorption spectrum of the complex, and interestingly the iminium pKa dropped to 6.0 with lower ΦESPT (entry 2). Table II-25. Spectroscopic and pKa change as the result of L77Y introduction. Entry hCRBPII Mutant a λabs λem λem ΦESPT pKa (Blue) (Red) 1 KLVSHWH:F16Y:L117C 520 577 680 95.5 8.6 2 KLVSHWH:F16Y:L117C:L77Y 502 571 670 41.5 6.0 a KLVSHWH equals to Q108K:K40L:T51V:T53S:R58H:Y19W:A33H. L77 is located on the loop at the entrance of the protein’s binding cavity (Figure 11-27a), and as previously Dr. Wenjing Wang showed for retinal/hCRBPII complexes, switching from a hydrophobic residue to more polar residues such as histidine, tyrosine, and cysteine could move the conformation of the loop so that it can flip out of the binding pocket to get more solvated in the aqueous solution. Such changes can affect the protein expression yield or its stability. For instance, in this study, none of the L77 mutations to serine or cysteine led to soluble proteins, 94 and for the expressed mutants, the formation of the aggregates was quite apparent from the UV-Vis spectrum of the corresponding complexes. Hence mutation of L77 was not continued in further studies. a. T29 L77 b. R58H 4.2 Å 4.8 Å A33H 2.1 Å 4.2 Å T29 4.3 Å 5.5 Å 4.4 Å 5.4 Å Figure II-27. a. Cartoon representation of Q108K:K40L:T51V:T53S:R58H:Y19W:A33H:F16Y:L117C/Thiophenol complex with T29 and L77 residues highlighted. b. Detailed hydrogen bonding network surrounding T29, A33H, R58H and the hydroxyl group in the same crystal. 95 II.11.3 B. T29 mutation T29 resides on the α-helices segment with its side chain pointing outside of the binding pocket to get better solvation of the polar hydroxyl group leading to a long distance (9.1Å) between threonine and the chromophore’s hydroxyl groups (Figure II-27). Hence, it is not expected for residues at position 29 to significantly affect the chromophore’s photoacidity. However, the crystal structure of Q108K:K40L:T51V:T53S:R58H:Y19W:A33H:F16Y:L117C/ThioPhenol complex reveals that there is a conserved water-mediated hydrogen bonding network including T29, A33H, the chromophore’s hydroxy group, and two organized water molecules (Figure II-27b). The well-developed network around the hydroxyl group plus the close-distanced R58H explains the high ESPT quantum efficiency with this mutant. We sought to examine whether mutation of T29 to tyrosine would influence the water network and/or the ΦESPT (Table II-26). Table II-26. Spectroscopic change as the result introduction of T29Y mutation. Entry hCRBPII Mutant a,b λabs λem λem ΦESPT Φ pKa (Blue) (Red) 1 KLVSHWHY:L117C 520 577 680 95.5 0.08 8.6 2 KLVSHWHY:L117C:T29Y 527 - 681 >99 0.13 8.9 a KLVSHWHY equals to Q108K:K40L:T51V:T53S:R58H:Y19W:A33H:F16Y. Interestingly, the introduction of T29Y led to an increase in both the ESPT and the red-shifted fluorescence efficiency as the ThioPhenol-PSB emission is almost completely suppressed for the corresponding complex (entry 2). Unfortunately, there is no crystal structure for this mutant to demonstrate the 96 orientation of T29Y, but presumably strong water-mediated hydrogen bonding with the chromophore is the reason for the enhanced fluorescence properties. T29Y mutation was then retained in the protein template for further engineering. II.11.3 C. T53 mutation Formerly, Dr. Wenjing Wang reported that T53C mutation could lead to a significant bathochromic shift in the absorption wavelength of retinal/hCRBPII complex due to the polarizability of the cysteine residue. Earlier in Section II.11.1, we described the effect of T53E on increasing the ΦESPT and bathochromic shifts in the emission of ThioPhenolate-PSB; however, this mutation was eliminated for further studies as the addition of L117C could not increase the iminium pKa and therefore mutants that contained T53E showed lower fluorescence quantum yield. We surmised that the polarizability of the T53C mutation could stabilize the excited state better than serine. Table II-27. Spectroscopic properties as the result of introduction of cysteine in positions T51 and T53. Entry hCRBPII Mutant a λabs λem λem ΦESPT Φ pKa (Blue) (Red) 1 T51V:T53S (M1) 527 - 681 >99 0.13 8.9 2 T51C:T53S (M2) 524 - 672 >99 0.11 9.4 3 T51V:T53C (M3) 517 - 680 >99 0.15 9.8 4 T51M:T53S (M4) 523 - 674 >99 0.12 8.8 a The mutations are introduced to Q108K:K40L:F16Y:Y19W:T29Y:A33H:T51V:T53S:R58H:L117C mutant. 97 In addition, in Section II.11.2, we showed that T51M mutation resulted in the PSB stabilization and increased pKa due to the same reasons. Thus, cysteine was introduced at positions 51 and 53 of the template (Q108K:K40L:F16Y:Y19W:T29Y:A33H:T51V:T53S:R58H:L117C), results in in the highest ΦESPT observed for this series (Table II-27). To our delight, as in the parent protein (entry 1), the blue emission of ThioPhenol-PSB is completely suppressed in all of the resultant mutants. The highest ΦESPT (>99.5%) was acquired upon the introduction of cysteine either at position 51 or 53. Although with T51C, the emission is slightly blue shifted. An interesting observation was made with T51C, as shown in Figure II-28a; there is a shoulder to the right side of the PSB absorption corresponding to the deprotonated hydroxy group of the chromophore, ThioPhenolate-PSB complex. As excitation of this small shoulder at neutral pH results in emission maximum at 673 nm, same as the ESPT product emission (Figure II-28b). The complex absorption was monitored via UV-Vis spectra upon both acid and base titration to ensure this claim is valid. Acidification of the solution leads to the disappearance of the shoulder (Figure II-28c). Upon basification of the sample, the intensity of the PSB absorption peak decreases while the shoulder grows, redshifting to 615 nm corresponding to ThioPhenolate-PSB. Upon basification to pH above 10, almost all of the PSB is deprotonated, and the absorption maximum around 400 nm corresponding to ThioPhenolate-SB appears (Figure II-28d). 98 These observations suggest that the shoulder is the deprotonated hydroxyl group in the ground state. a. b. ThioPhenolate- PSB c. d. PSB- OH SB-O PSB-OH SB-OH PSB-O PSB-O Figure II-28. a and b. The absorption and emission spectra of Q108K:K40L:F16Y:Y19W:T29Y:A33H:T51C:T53S:R58H:L117C/ThioPhenol complex. The shoulder corresponds to PSB-Phenol complex since excitation of both results to the same emission. c. The acid titration and d. the base titration of the same complex. 99 Although the hydroxyl group’s pKa value is quite high, in the range of 9.2 to 10.5 depending on the protein mutant, it can partially get deprotonated at neutral pH in the ground state upon binding to protein mutants that have a well-developed water-mediated hydrogen bonding network. Nonetheless, the formation of ThioPhenolate-PSB in the ground state is not desired in this study as we are focused on developing far-red/NIR-emitting tags through the ESPT of a photoacid. PSB-O Figure II-29. Comparison of absorption (left) and emission (right) of M1 (T51VT:53S), M2 (T51C:T53S), M3 (T51V:T53C), and M4 (T51M:T53S) protein/ThioPhenol complexes. As listed in Table II-27, all four protein mutants exhibit the ΦESPT (>99%) and high iminium pKa values (8.8-9.8). However, the highest fluorescence quantum efficiency was acquired with M3/ThioPhenol complex (entry 3). In addition, comparing the absorption and emission spectra of these mutants shows that the shoulder corresponding to the unwanted ThioPhenolate-PSB complex is 100 the smallest with M3 (Figure II-29). Thus, this mutant was chosen for live-cell imaging experiments. II-12 ThioPhenol/hCRBPII binding kinetics To investigate the proper staining duration, binding kinetics were measured prior to the confocal imaging experiments. 20 µM of the protein in PBS buffer at neutral pH (7.2-7.4) were incubated with 0.5 equivalent of the ligand at 23 °C and increase in absorbance of the corresponding PSB at its 𝜆max was recorded. Collected data points were fit with a second-order rate equation considering multiple reagents protein and the ligand with non-equal concentrations (Figure II- 30, see section IV.3.4 for detailed fitting description). PSB Formation k = 1467 M-1.min-1 t1/2 = 34.1 min R2 = 0.999 Figure II-30. Rate of ThioPhenol/M3 PSB formation, fitted to 2nd order kinetics with 20 μM protein and 10 μM Thiophenol. Plotted is the concentration of free chromophore vs. time. The next section of this chapter will demonstrate this probe’s usefulness in live-cell imaging; most importantly, ThioPhenol cell permeability and its efficient 101 target binding are shown. As was predicted, acquired confocal images display no fluorescent background even with overnight incubation with the ligand and without any washing steps prior to imaging. II.13 Visualization of hCRBPII/ThioPhenol in mammalian cells Next, the performance of engineered hCRBPII/ThioPhenol complexes as a no-wash live-cell imaging system was investigated. The mutant Q108K:K40L:F16Y:Y19W:T29Y:A33H:T51V:T53C:R58H:L117C (M3) was chosen for these studies. The iminium pKa of this mutant is high (9.85), resulting in predominant PSB formation, the ΦESPT (>99%) and fluorescence quantum yield (0.15) is the highest among all mutants with, and moreover, the concentration of ThioPhenolate-PSB in the ground state was negligible with M3 (Figure II-31). 517 nm 680 nm pKa Titration pKa= 9.85 R2 = 0.999 Figure II-31. Spectroscopic properties of Q108K:K40L:F16Y:Y19W:T29Y:A33H:T51V:T53C:R58H:L117C mutant with ThioPhenol including UV-Vis and fluorescence spectra (left) and pKa titration (right). 102 Additionally, binding was relatively fast; the second-order half-life for iminium formation is 34 minutes at 23 °C, and it is solely expressed in the monomeric form (Figure II-30). All spectroscopic properties of ThioPhenol/M3 are summarized in (Table II-28). Table II-28. Spectroscopic properties of ThioPhenol/M3 complex. e k t1/2 b Complex λabs λem ΦESPT Φa pKa (M-1.cm-1) (M-1.min-1) (min) ThioPhenol/M3 517 679 33,743 >99 0.15 9.8 1467 34 a Absolute quantum yield was measured on a Quantaurus-QY. b Half-life based on the rate constant obtained from second order rate fitting; measured at 23 °C with 20 μM protein and 0.5 equiv ThioPhenol at pH 7.2. II.13.1 ThioPhenol/M3 successfully labels hCRBPII in HeLa cells To test the performance of ThioPhenol as a no-wash tag for in vivo imaging experiments, M3-hCRBPII was cloned into the pFlag-CMV2 vector containing EGFP fused on the N-terminus of hCRBPII to label the whole cell. EGFP pFlag-CMV2- EGFP-hCRBPII-SP hCRBPII AmpR Signaling Peptide Figure II-32. Maps of the EGFP-hCRBPII-SP fusion constructs. SP: signaling peptides. SP = 3×NLS (nuclear localization sequence), NES (nuclear export sequence), and CAAX (prenylation tag). 103 Additionally, to target M3 at cell nuclei, cytosol, or plasma membranes, the signaling peptides NLS (nuclear localization sequence), NES (nuclear export sequence), and CAAX (prenylation tag) were fused to the C-terminus of M3, respectively as illustrated in Figure II-32. The fused constructs were transfected and expressed in HeLa cancer cell lines to test the applicability of ThioPhenol. Imaging was performed by incubating HeLa cells with 10 μM ThioPhenol for one and half hours at 37 °C. The cells were then directly subjected to the confocal imaging without any washing steps prior to imaging. In all cases, the green fluorescence from EGFP was observed when excited at 488 nm, indicating that transfection was successful, and the fusion protein has been expressed (Figures II-33 and II-34, the green channel). To collect the NIR emission, stained cells were excited at 514 nm. In every triple-fused construct, the NIR emission 620 nm-720 nm window) demonstrated the same pixel specificity as that of EGFP, confirming that the NIR fluorescence is purely emitted from the activated ThioPhenol/M3 complex without any signal contamination from non- specific labeling that results in identical images in the green and red channel (Figures II-33 and II-34, the red channel). Apparently, no fluorescence background is observed in the red channel, indicating that Thiophenol does not label off-target lysines or non-specific bindings does not lead to fluorescence signal. These results prove the utility of the no-wash labeling of 104 Q108K:K40L:F16Y:Y19W:T29Y:A33H:T51V:T53C:R58H:L117C with ThioPhenol in mammalian cells. Furthermore, as discussed above, size exclusion chromatography results confirmed that M3 is expressed solely in monomeric form (up to 19 mg/L). This is advantageous, assuming the monomeric state dominates in cellular environments as well. To ensure that labeling with ThioPhenol does not develop fluorescent background over long incubation times, HeLa cell lines were incubated with 10 µM ThioPhenol at 37 °C for about 12-15 h and were imaged without washing steps. Notably, no non-specific fluorescent background was observed even after overnight incubation time, proving the selectivity of fluorescent signal of ThioPhenol/M3-hCRBPII tag. II.14 Conclusion and future research directions As stated at the beginning of this chapter, developing fluorogenic background-free tags is one of the most important goals of our research. This chapter describes how this goal is achieved by coupling a non-fluorescent chromophore with various hCRBPII mutants. In this designed system, the protein is engineered to maintain a high iminium pKa binding to the non-emissive chromophore as a PSB. Further modification of the protein host (hCRBPII) via point mutagenesis makes it possible to activate the complex to function as a photoacid and generate a NIR fluorescence signal. The strong ICT system formed between the alkoxide and the iminium in the ThioPhenolate-PSB complex, upon 105 photoirradiation results in a bathochromically-shifted emission. No fluorescent background signal was observed as a result of non-specific imine or iminium formation in the cellular milieu. This is due to the high pKa of the phenol moiety (9.5-10), which cannot be deprotonated in non-target hosts. Also, the NIR emission of the ThioPhenolate-PSB is well separated from unbound chromophore or non- specific bindings emission. The fluorogenic characteristics of this system make it useful in no-wash background-free NIR imaging applications, as demonstrated by the ThioPhenol/M3 complex, which successfully labeled HeLa cell lines without any fluorescent background. Our next goal is to modify the ThioPhenol structure to increase the quantum yield and, ultimately, the brightness of the tags. ThioPhenol structure optimization and rigidifying its structure to reduce the non-radiative relaxations of the excited state are discussed in Chapter III. In addition, extending the application of this system in two-color or multi-color imaging is followed and described in the next chapter. 106 Nucleus localization EGFP-3NLS ThioPhenol/hCRBPII-3NLS Ex: 488 nm Ex: 515 nm Em: BP 500 - 550 nm Em: BP 620 - 720 nm EGFP-3NLS + DIC ThioPhenol/hCRBPII-3NLS + DIC Figure II-33. Confocal imaging of labeled HeLa cells expressing EGFP-hCRBPII- 3NLS. NLS = nuclear localization sequence. Cells were stained with 10 µM ThioPhenol and incubated at 37 °C for 1 h and 30 min. Cells were not washed before imaging. 107 Nucleolus export localization EGFP-NES ThioPhenol/hCRBPII-3NES Ex: 488 nm Ex: 515 nm Em: BP 500 - 550 nm Em: BP 620 - 720 nm Green + Red Channel DIC Figure II-34. 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Journal of Biological Chemistry 284, 13285-13289, doi:10.1074/jbc.R800080200 (2009). 121 CHAPTER III: DOUBLE EXCITED-STATE PROTON TRANSFER PHENOMENON: DEVELOPING PROTEIN TAGS WITH APPLICATIONS IN NO-WASH FLUORESCENT IMAGING Proton-transfer reactions remain one of the most fundamental and vital processes in chemistry and biology.1-3 Excited-state proton transfer (ESPT) plays a crucial role in many biological systems, such as photosystem II,1,4,5 DNA,6,7 bacteriorhodopsin,8,9 and green fluorescent protein.10,11 Given the importance of ESPT processes and its role as a functional tool in different areas such as fluorescent probes,12-14 white light-emitting materials,15,16 biological imaging,17,18 pH jumps,19,20 and triggers for protein folding,21 a large number of experimental and theoretical studies have been focused on these systems to investigate the underlying dynamics and principles. Photoacids and photobases are a class of molecular systems that exhibit a profound increase in acidity or basicity, respectively, upon photoexcitation into the first excited electronic state. As described in Chapter II, photoacidity is a reversible process as relaxation to the ground state changes the molecule back to the low- acidity state. Photoacids have been studied since the 1970s,22-25 and they have found profound application in organic optoelectronic materials,26,27 dye-sensitized ion exchange membranes,28,29 large Stokes shift fluorescent proteins,30,31 and to control molecular and supramolecular events. Generally, the ground state pKa values range from 5 to 10, and the ΔpKa (the difference between the ground and excited-state pKa values) ranges from 3 to 13.32 122 As mentioned earlier, during the ESPT process, the chromophore moves along a proton to the protein through a proton wire inside the protein cavity. This phenomenon has been found in the Large Stokes Shift (LSS) red fluorescent proteins such as LSSmKate1 and LSSmKate2.33-35 Structural analysis of other examples of ESPT-capable GFP variants, including mKeima, LSSmOrange, and mBeRFP, shows that the active site of these proteins can also be optimized to provide analogous proton relays to red-shift their emission. In particular, based on these predictions and rational engineering of the immediate chromophore environment, Piatkevich and coworkers produced LSS variants of several conventional orange and red FPs, including mCherry, mNeptune, mStrawberry, mOrange, and mKO through generating proton wires around the chromophore.33 Unlike photoacids, there are only a few reports of photobases in the literature, which are limited to heterocyclic amines such as acridines,36,37 aminoanthraquinones,38 3-styrylpyridines,39 Schiff bases,40 and quinolines.41 These photobases are distinct from photobase generators (PBGs) generated upon light irradiation of their salts, which have their pitfalls of being irreversible with slow proton transfer rates.42 The paucity of photobases capable of generating more basic species upon photoirradiation has hindered their exploitation. Nevertheless, the ability to control the basicity of a compound upon photoexcitation is as important as those exhibited by photoacids and open to discovery. 123 III.1 Previous work toward developing large Stokes shift fluorescent protein- based photobases As discussed previously, most ESPT-capable FPs are photoacids, wherein fluorescence is activated through proton transfer to the nearby residues upon excitation. A complementary approach would be a photobase in which photoirradiation leads to a more basic species capable of proton abstraction that leads to the generation of a cationic system. It was envisaged that a protein/chromophore complex with photobasic properties is capable of intramolecular charge transfer (ICT). If the photogenerated cation resides in conjugation with a polyene that is terminated with an electron-donating substituent, the photoactivation will lead to an electronic ‘push-pull’ system typical of large Stokes shift bathochromic pigments with ICT characteristics (Figure III-1). hν hν A A* A A* B* SS LSS Abs/Em Abs/Em Wavelength (nm) Wavelength (nm) Standard Fluorescence Fluorescence from an Altered Excited State Figure III-1. Model for a large Stoke shift (LSS) vs. a standard fluorescence system. Large Stokes shift red-shifted emission results via conversion of the excited state species A* to a bathochromically distinct molecule B*. 124 The inspiration to design such a system was our recent discovery. Dr. Wei Sheng reported the photobasic behavior and proton transfer dynamics of a fluorene-based imine, FR0-SB, a conjugated Schiff Base (SB) obtained upon the imine formation between the aldehydic form of FR0 dye with n-butylamine.43 The huge change in this system’s basicity, 14 unit increase in pKa, upon photoirradiation enables proton abstraction from protic solvents in its excited state, which generates the subsequent iminium that emit with over 200 nm apparent Stokes shift (Figure III-2).43 hv a. n-BuNH2 H N N N O N C 4H 9 N C 4H 9 H FR0 FR0-SB FR0-PSB λabs = 395 nm λem = 554 nm b. 372 nm 488 nm 628 nm 463 nm Figure III-2. a. FR0 structure and formation of FR0-SB and FR0-PSB. b. UV-Vis (left) and fluorescence (right) spectra of FR0-SB (blue) and FR0-PSB (red) in ethanol. 125 Excitation of FR0-SB at its maximum absorption wavelength (372 nm), when dissolved in protic solvents, results in dual emission bands with maxima at 463 nm and 628 nm corresponding to FR0-SB and FR0-PSB complexes, respectively. This assignment was confirmed as the excitation of the FR0-PSB, which can be produced upon acidification of the SB sample, gives rise to the same, red-shifted emission at about 628 nm (Figure III-2). This discovery encouraged us to design fluorescent tags produced upon incorporating a fluorophoric photobase into a rationally engineered protein carrier to create photoactivatable LSS-FPs. Recently, Dr. Elizabeth Santos and Dr. Wei Sheng successfully designed protein-based photobases capable of generating highly conjugated polar ICT systems in the excited state that are well red-shifted in emission and show high fluorescence quantum efficiency. In their designed system, the fluorophore covalently binds hCRBPII through the reaction of the aldehydic ligands with an active site lysine residue. Additionally, the protein needs to maintain a low iminium pKa to bind the fluorophore as an imine. The appropriate positioning of acidic amino acid sidechains is also critical in this study as they facilitate the proton transfer to the imine upon excitation and subsequent iminium generation. Although FR0 was the first ligand that showed photobasic properties, its strong activity as an ESPT-capable fluorophore is detrimental in achieving selectivity, and therefore, its application as a low background imaging tag is significantly hampered. On the contrary, the imine of ThioFluor and FR1 126 (derivative of FR0) exhibited weak ESPT in protic solvents (Figure III-3b), which is essential for cell imaging since then the iminium is only generated in the binding cavity of the engineered target protein. a. ThioFluor-SB ThioFluor-PSB O !abs = 400 nm !abs = 521 nm S !em = 540 nm !em = 689 nm Me2N Φ = 7% Φ = 2% ThioFluor n-BuNH2 H N hν, ROH N Bu Bu FR1-SB FR1-PSB Et2N !abs = 394 nm !abs = 510 nm !em = 510 nm !em = 690 nm FR1 O Φ = 5% Φ = 2% b. Figure III-3. a. Formation of the imine and iminium of ThioFluor and FR1 in ethanol. b. Spectroscopic properties of FR0 (blue), FR1 (red), and ThioFluor (green) in ethanol. UV-Vis (left) and fluorescence spectra upon SB excitation (right). Same as FR0, the selected ligands, ThioFluor and FR1 (a derivative of FR0), provide the structural requirements for generating an ICT system; an 127 alkylamino group as the electron-donor, a p-spacer, and the imine unit as the electron-withdrawing group. The formation of the corresponding iminium upon the ESPT process results in a strong push-pull system, leading to an apparent large Stokes shift over 200 nm (Figure III-3a). After extensive experiments, K40 was recognized as the best position to introduce acidic residues as the proton source for the ESPT process. We arrived at the Q108K:K40E:T53A:R58L:Q38F:Q4F-hCRBPII (M3) mutant exhibiting the highest values for ΦESPT. As described in Chapter II, we defined ΦESPT as the fraction of total fluorescence originating from the excited state iminium. a. b. 394 nm 605 nm 380 474 SS > 200 SS < 100 Figure III-4. UV-Vis and fluorescence spectra of M3/ThioFluor complex (394 nm excitation), exhibiting ESPT emission (left) vs. UV-Vis and fluorescence spectra of M1/ThioFluor complex (380 nm excitation) leads to SB emission at 474 nm (right). The spectra have been measured in PBS buffer at neutral pH (7.2). Comparing the absorption and emission spectra of this mutant with that of Q108K:K40L:T53A:R58L:Q38F:Q4F-hCRBPII (M1) /ThioFluor complex in which 128 the acidic residue at position 40 (40E) is substituted with a neutral amino acid (40L) confirms that the ESPT originates from K40E mutation to the imine. As the emission wavelength is blue-shifted by about 130 nm and the Stokes shift is much smaller with the K40L mutant, which is an indication of the emission from the imine and not the iminium (Figure III-4). M3 was selected for live-cell fluorescent imaging experiments. It shows low iminium pKa values (~5.2); the Q4F mutation typically increases soluble protein expression, but more importantly, it helps to suppress the iminium pKa leading to more imine formation in the ground state. Moreover, the three hydrophobic mutations, T53A, R58L, and Q38F, were introduced to increase binding affinity and enhance the rate of chromophore binding. The binding kinetics are also fast with this mutant (the binding is complete in less than 5 minutes) compared to non- specific imine formations, which is significantly important in increasing the selectivity for imaging the desired target. EGFP-M3-NES M3/FR1 complex-NES Figure III-5. M3/FR1 imaging in live HeLa cells. NES = nuclear export sequence. Cells were stained with 500 nM FR1 and incubated at 37 °C for 1 min. Cells were washed three times with DPBS before imaging. Scale bar, 10 𝜇m. 129 Notably, using such a strategy to produce fluorescent tags with minimum fluorescence background is unique and unprecedented. Next, we chose to show the application of the photobasic complexes in live-cell imaging (Figure III-5). The required condition for cell imaging with FR1 is one-minute incubation (500 nM) HeLa cells at 37 °C, followed by three washing steps with DPBS to remove unbound FR1. However, imaging experiments with ThioFluor complexes were not as successful since residual free ligand led to fluorescence background. III.2 Designing hCRBPII/dye complexes that undergo double ESPT upon a single excitation Chapter II described the photoacidic properties and excited-state proton transfer dynamics of ThioPhenol in complexation with hCRBPII. ThioPhenol as a photoacid emits in the far-red/NIR region of the spectrum (~700 nm) with a large Stokes shift of about 150 nm. We were able to develop a no-wash live-cell imaging using this system as the free ThioPhenol is unable to form a push-pull system and is not emissive. Furthermore, since the phenol moiety of the chromophore has a high pKa, its deprotonation is only feasible in an engineered hCRBPII mutant leading to a substantial decrease in fluorescence background originating from non- specific imine or iminium formation. Developing protein-based fluorescent tags with minimum background has been one of our lab’s overarching goals. Successful no-wash background-free imaging experiments with ThioPhenol inspired us to explore its properties as a photobase. In addition, we sought to investigate whether ThioPhenol complexes 130 can be developed as a system that can show both photobasic and photoacidic characteristics upon a single excitation. III.2.1 General protein host properties required for a double ESPT system In order to develop the photobase part of the complex, the protein should be able to maintain a low iminium pKa, so the imine is the only species formed in the ground state. At the same time, the mutations that helped deprotonate the phenol moiety should be retained to enhance photoacidic properties. a. Photobase H N Double ESPT N Photoacid S hν HO S O ThioPhenol-SB ThioPhenolate-PSB H b. N S O ICT Figure III-6. a. Formation of ThioPhenolate-PSB complex through a double ESPT process. b. The internal charge transfer resulted from the ThioPhenolate-PSB complex, the product of the double ESPT process. ThioPhenol binds the engineered protein as the ThioPhenol-SB and converts to ThioPhenolate-PSB through a double ESPT process in our proposed system (Figure III-6a). During this process, the SB grabs a proton from its vicinity and forms a Protonated Schiff Base (PSB), and simultaneously the phenol moiety loses a proton and generates the negatively charged oxygen (phenolate). The 131 excited state complex, ThioPhenolate-PSB, possesses a strong D−p−A and thus the resultant ICT leads to fluorescence (Figure III-6b). III.3 ThioPhenol-hCRBPII as a photobasic system As discussed in Section III-1, we recently discovered that glutamic acid at position 40 is the key residue in photobasic hCRBPII/fluorophore complexes that provides the proton for the ESPT process. The highest ΦESPT value was obtained with Q108K:K40E:T53A:R58L:Q38F:Q4F(M3)-hCRBPII mutant (>99%). We sought to investigate whether ThioPhenol/M3 complex could provide the same results in the first step. For all measurements, 20 µM of protein is incubated with 0.5 equivalent of the ligands in PBS buffer at pH 7.2 (Table III-1). Table III-1. Spectroscopic properties in complexation with M3. Entry Ligand λabs λem SS ΦESPT Φa pKa 1 FR1 392 595 203 > 0.99 0.72 5.2 2 ThioFluor 397 605 208 > 0.99 0.51 5.1 5 ThioPhenol 373 534 161 > 0.99 0.05 <5 a Absolute quantum yield was measured on a Quantaurus-QY. Both absorption and emission wavelengths are blue-shifted with ThioPhenol compared to the other ligands, about 21 nm and 60 nm, respectively. Due to the weak electron-donating properties of the hydroxyl group, ThioPhenol is unable to form a strong ICT system, which is effective in producing larger Stokes 132 shifts and red-shifted emission. Another outcome of the weak ICT characteristic of the ThioPhenol/M3 complex is its decreased fluorescence quantum efficiency. As shown in Table III-1, the emission wavelength with ThioPhenol is not as red-shifted as with the other ligands; however, emitting at 534 nm verifies the PSB formation upon excitation. Nonetheless, pH titration of the complex and monitoring its UV-Vis and emission spectra illustrates that the emission originates from the PSB. UV-Vis and emission spectrum at 373 nm. Figure III-7. UV-Vis, emission spectra of Q108K:K40E:T53A:R58L:Q38F:Q4F/ThioPhenol complex. The absorption and emission spectra of the ThioPhenol/M3 complex measured at neutral pH 7.4 are shown in Figure III-7. The lower iminium pKa of this complex eliminates the residual PSB formation observed with ThioFluor and FR1. The acidification of the complex solution leads to the PSB formation absorbing at 488 nm; the excitation of PSB gives rise to the emission at 571 nm, which is 37 nm more red-shifted than the PSB produced upon excitation of the SB 133 (Figure III-8a). Such a trend in emission wavelength of PSB was observed with ThioFlour as well. A red-shift of 25 nm is observed for the emission wavelength of the PSB produced upon acidification of the sample compared to the PSB as the ESPT product (Figure III-8b). a. b. Figure III-8. a. UV-Vis (left) and fluorescence (right) spectra of Q108K:K40E:T53A:R58L:Q38F:Q4F/ThioPhenol complex upon acidification of the solution. b. UV-Vis (left) and fluorescence (right) spectra of Q108K:K40D:T53A:R58L:Q38F:Q4F/ThioFluor complex upon acidification of the solution. 134 The ThioPhenol/M3 complex was stable enough upon basification of the sample to a pH value of more than 10. The absorption wavelength slightly red- shifts (about 10 nm) as the hydroxyl group becomes deprotonated and the ThioPhenolate-SB complex forms. An interesting observation was made in this study as the excitation of SB even at pH 9.2 results in the same emission collected at neutral pH but with lower intensity, which is surprising since in such a basic pH, glutamic acid is not expected to act as a proton source for the ESPT process (Figure III-9). Figure III-9. UV-Vis (left) and fluorescence (right) spectra of Q108K:K40E:T53A:R58L:Q38F:Q4F/ThioPhenol complex upon basification of the solution. To further study ThioPhenol complexes as a photobase, we chose to measure the photophysical properties in conjugation with Q108K:K40L:T53A:R58L:Q38F:Q4F (M1) mutant wherein the glutamic acid at position 40, the proton source, is substituted with leucine. The resultant complex, ThioPhenol/M1, shows a low iminium pKa (5.1), leading to SB formation only. 135 However, surprisingly, in contrast to ThioFluor/M1, excitation of the SB at 370 nm led to the PSB emission at 531 nm (Table III-2), the same as what was collected with M3 (see Figure III-4b and Figure III-2). Unfortunately, we were unable to obtain a crystal structure to investigate the possibility of other proton sources. Table III-2. Spectroscopic properties in complexation with M1. Entry Ligand λabs λem (SB) λem (PSB) SS ΦESPT pKa 1 ThioFluor 379 474 – 95 < 0.02 5.1 2 ThioPhenol 370 – 532 162 0.97 <5 III.4 Developing ThioPhenol-hCRBPII complexes showing both photoacidic and photobasic characteristics In Chapter II gradual evolution of ThioPhenol-hCRBPII complexes as a photoacidic system was elaborated; photoirradiation of the complex led to deprotonation of the chromophore’s hydroxyl group and subsequent NIR emission with a large Stokes shift. Additionally, the successful application of ThioPhenol- SB as a protein-based photobase was discussed in the previous section. Thus, we sought to investigate engineering hCRBPII into a new class of fluorescent proteins that exhibits both photoacidic and photobasic properties upon a single excitation. As shown in Section II-7, R58H is an essential mutation in deprotonating the hydroxyl group; therefore, leucine in M3 was substituted with histidine, and the photophysical properties of the resultant complex (Q108K:K40E:T53A:R58H:Q38F:Q4F-hCRBPII (M4)/ThioPhenol) were obtained. ThioPhenol binds M4 solely as imine with a maximum absorption wavelength of 136 370 nm. Gratifyingly, excitation of the SB results in an unprecedently large Stokes shift ~ 242 nm indicative of the double ESPT processes. To our delight, the ΦDESPT (the quantum yield of double ESPT process) is more than 99% resulting in a well separated absorption and emission spectra and minimum self-absorption (Figure III-10). ΦDESPT is defined as the fraction of total fluorescence from the excited state ThioPhenolate-PSB to the total fluorescence (see Section II-7 for detailed description). 370 nm 612 nm SS ~ 250 Figure III-10. Normalized absorption and emission spectra of Q108K:K40E:T53A:R58H:Q38F:Q4F/ThioPhenol complex, when excited at 370 nm at neutral pH (~7.2). As proposed in Section III-2, two ESPT processes happen upon photoirradiation of this system. Presumably, the hydroxyl group acts as a photoacid losing a proton to R58H. At the same time, the SB segment acts as a photobase and grabs a proton resulting in PSB formation (see Figure III-6). In fact, the products of single and double ESPT processes are the same: ThioPhenolate- 137 PSB complex and the hydroxyl group becomes deprotonated in both designs. The only difference is that for the single ESPT processes, the iminium pKa value is engineered to be high enough (~10), leading to a ground state PSB. While for dual proton transfer systems, the chromophore binds as a SB due to the low iminium pKa (<5) and converts to the PSB upon photoexcitation (Figure III-11). pKa ∼ 10 H H N N S Single ESPT S HO O ThioPhenol-PSB ThioPhenolate-PSB pKa < 5 H N N S Double ESPT HO S O ThioPhenol-SB ThioPhenolate-PSB Figure III-11. Schematic representations of single and double ESPT processes. The starting complex is different, while the product is the same. As described earlier, ThioPhenol photobasic complexes’ quantum efficiency is low due to the weak electron-donating effect of the hydroxyl group. Therefore, enhanced brightness was expected for the double ESPT complexes as the Hammett value for the alkoxide group is much higher and almost the same as the N,N dimethyl amino group. The photophysical properties of ThioPhenol/M3 and M4 are compared in Table III-3. Both mutants show the same imine absorption wavelength; however, excitation of ThioPhenol/M4 imine at 370 nm results in 80 nm bathochromic shifts 138 in the emission wavelength compared to ThioPhenol/M3 complex. Notably, the fluorescence quantum efficiency with M4 is increased to 16% due to the strong ICT system formation upon excitation. Table III-3. Spectroscopic properties of ThioPhenol/M3 and M4 complexes. complexation with M1. Entry hCRBPII mutant λabs λem SS ΦESPT Φa M3 Q108K:K40E:T53A:R58L:Q38F:Q4F 370 532 162 0.97 0.05 M4 Q108K:K40E:T53A:R58H:Q38F:Q4F 370 612 242 0.98 0.16 a Absolute quantum yield was measured on a Quantaurus-QY. To ensure that the red-shifted emission of the ThioPhenol/M4 complex is the result of a double ESPT process, the UV-Vis and fluorescence spectra of this complex were monitored upon acid and base titration of the sample. a. b. 370 nm 612 nm 491 nm 570 nm Figure III-12. a. UV-Vis spectra of Q108K:K40E:T53A:R58H:Q38F:Q4F/ThioPhenol complex upon acid titration of the sample. b. Fluorescence spectra of the same complex upon SB excitation at neutral pH and PSB excitation in acidic pH. 139 The PSB absorption peak at 491 nm starts to appear upon acidification of the complex solution to pH < 6. Putatively, the SB absorption decreases; however, it never disappears due to the low pKa (Figure III-12a). PSB excitation at pH 4.7 leads to an emission wavelength at 570 nm, more than 40 nm blue-shifted when excited at SB, proving that the emission peak at 612 is due to the ThioPhenolate- PSB complex formation (Figure III-12b). Interestingly, the double ESPT process happens even at pH values up to 9.1. Excitation of the SB at pH more than 10 leads to a dual emission at 507 and 612 nm, which originates from the ThioPhenolate-SB and the ThioPhenolate PSB, respectively (Figure III-13). III.5 Attempts to increase the fluorescence quantum efficiency of DESPT derived emission PSB-O PSB-O Figure III-13. UV-Vis spectra of Q108K:K40E:T53A:R58H:Q38F:Q4F/ThioPhenol complex upon base titration (left). Fluorescence spectra of the same complex upon SB excitation in different pH. 140 In our first attempt to generate a double ESPT system that shows both photoacidic and photobasic properties, R58H mutation, recognized as the key residue in the hydroxyl group deprotonation, was added to the photobasic protein mutant. The excitation of the SB in the corresponding complex, ThioPhenol/M4, results in an emission spectrum, in which its maximum wavelength is even more red-shifted than the PSB emission. A large Stokes shift (~250 nm) and high quantum yield (16%) of the red-shifted emission were acquired with this single mutation. III.5.1 Expanding the water-mediated hydrogen bonding network around the hydroxyl group Table III-4. Spectroscopic properties upon addition of F16Y, A33H and T29Y. Entry hCRBPII Mutant a λabs λem SS ΦDESPT Φ (Red) 1 KEAFF:R58H:F16Y 370 594 224 > 0.99 0.16 2 KEAFF:R58H:A33H 375 613 238 0.98 0.15 3 KEAFF:R58H:F16Y:A33H 373 596 223 > 0.99 0.15 4 KEAFF:R58H:Y19W:A33H 370 614 244 > 0.99 0.14 5 KEAFF:R58H:T29Y 371 614 243 > 0.99 0.16 6 KEAFF:R58H:F16Y:T29Y 371 594 223 > 0.99 0.14 a KEAFF equals to Q108K:K40E:T53A:Q38F:Q4F. As described in Chapter II, a few residues have played a critical role in expanding the hydrogen bonding network and consequently enhancing the ΦESPT. We sought to investigate whether the addition of those residues such as F16Y, A33H, T29Y, and their combinations could increase the fluorescence quantum 141 yield (Table III-4). The SB was the only species formed in neutral pH for all mutations due to the low iminium pKa (in the range of <5 to 5.2). Interestingly, all mutants show excellent DESPT characteristics with none showing single ESPT emission around 530 nm (imine to iminium). The ΦDESPT was more than 97% for all mutants. Noteworthy, mutants with F16Y mutation showed about 20 nm blue- shift in their emission wavelength (entries 1, 3, and 6), which is presumably due to the interaction of the phenol moiety and the tyrosine residue at position 16, leading to localization of the resultant phenoxide negative charge in the excited state (see Section II-10). Table III-5. Spectroscopic properties as the result of substituting 58H with 58Y. Entry hCRBPII Mutant a λabs λem SS ΦDESPT Φ (Red) 1 KEAFF:R58H:F16Y 370 594 224 > 0.99 0.16 2 KEAFF:R58Y:F16Y 363 535 137 0.13 0.07 a KEAFF equals to Q108K:K40E:T53A:Q38F:Q4F. To ensure that R58H is the essential residue for the dual proton transfer process, histidine at position 58 was substituted with tyrosine, another proton acceptor residue in the Q108K:K40E:T53A:Q38F:Q4F:R58H:F16Y template. The photophysical properties of the resultant mutant are shown in Table III-5. Interestingly, changing the histidine to tyrosine results in a broad peak maximized at 534 nm, the same as the emission wavelength of photobasic complexes such as ThioPhenol/M3. As a result, the ΦDESPT decreased to less than 10%. These 142 results show that R58H mutation is vital in the double ESPT process; thus, this mutation is retained in all further protein engineering for this study. In the course of engineering hCRBPII as a protein host capable of activating the photoacidity of ThioPhenol, we learned that methionine at position 51 helps to increase the ΦESPT (see Section II-11.2 B). Hence, we sought to explore the effect of this mutation on the fluorescence quantum efficiency of the double ESPT derived emission (Table III-6, entries 1 and 2). Unfortunately, there is no crystal structure available for double ESPT proteins. However, overlaying different ThioPhenol crystal structures with ThioFluor shows that in the same protein mutant, the orientation of these two structures is similar. Even with a different iminium configuration, the residues surrounding the end of the chromophore are almost in the same orientation (see Figure II-22) Hence, with this assumption in mind, we decided to examine close residues to the nitrogen atom of the dimethylamino moiety in the crystal structure of Q108K:K40E:T53A:R58W:Q38F:Q4F:Y19W/ThioFlour complex (Figure III- 14). As shown in Figure III-14, F16, M20, A33, S76, and L77 are the closest residues to the nitrogen atom. However, our previous studies had demonstrated that the mutation of S76 is not well tolerated, and thus, this residue remained unchanged. Due to their close distance to the hydroxyl group, F16Y and A33H are the two key mutations that enhanced the ΦESPT and photoacid single ESPT emission quantum yield; however, the addition of these mutations to M4 did not 143 result in improved photophysical properties (Table III-4). Hence, we chose to explore the effect of other proton acceptor residues at these positions on the double ESPT fluorescence quantum yield (Table III-6, 5, 6, and 7). 5.2 Å F16 M20 A33 5.3 Å 5.0 Å Q108K 4.1 Å 4.6 Å L77 S76 Figure III-14. The crystal structure of Q108K:K40E:T53A:R58W:Q38F:Q4F:Y19W/ThioFlour and the closest residues to the nitrogen atom of the dimethyl amino moiety of ThioFluor. 144 Additionally, M20 and L77 are in relatively close vicinity of the hydroxyl group, and thus several proton acceptors such as methionine, cysteine, and tyrosine were inserted at these positions. Unfortunately, most of these mutants did not yield soluble protein expressions. The photophysical properties of those that did express are listed in Table III-6, entries 2, 3, 8, and 9. Table III-6. Spectroscopic properties of R58H containing mutants. Entry hCRBPII Mutant a λabs λem SS ΦDESPT Φ 1 KEAFF:R58H:T51M 375 611 236 0.97 0.07 2 KEAFF:R58H:F16Y:T51M 370 583 213 > 0.99 0.16 3 KEAFFQ:R58H:L77M 370 542/609 172/239 0.51 0.10 4 KEAFF:R58H:F16Y:L77M 370 596 226 > 0.99 0.12 5 KEAFF:R58H:A33M 375 539/608 164/233 0.47 0.05 6 KEAFF:R58H:F16Y:A33M 375 592 217 0.97 0.09 7 KEAFF:R58H:F16Y:A33C 375 595 220 > 0.99 0.14 8 KEAFF:R58H:M20Y 370 613 243 0.96 0.10 9 KEAFF:R58H:F16Y:M20Yb 380 545 165 0.36 0.04 a KEAFF equals to Q108K:K40E:T53A:Q38F:Q4F. b This mutant shows a higher iminium pKa. The addition of T51M mutation reduced the fluorescence quantum yield to 7% (entry 1), however in combination with F16Y mutation, it recovered to 16% same as for the M4 complex (entry 2). Nonetheless. the ΦDESPT is more than 0.95 for both mutants. Interestingly, the single addition of L77M mutation results in a doubly maximized fluorescence spectrum around 542 nm and 609 nm, corresponding to photobase single ESPT and doubles ESPT emission (Figure III- 145 15). Again, better results are acquired upon the addition of both F16Y and L77M mutation (entries 3 and 4). Photobase single ESPT Double ESPT H H N hν N N S S S HO + O HO ThioPhenol-SB ThioPhenol-PSB ThioPhenolate-PSB Figure III-15. Products of single ESPT and double ESPT processes. Absorption and emission spectrum of Q108K:K40E:T53A:Q38F:Q4F:R58H:L77M/ThioPhenol upon SB excitation at 370 nm. The ΦDESPT dramatically decreased upon the addition A33M mutation (entry 7). The excitation of the SB at 375 nm led to an emission spectrum maximized at 539 nm with a shoulder at 608 nm that can be characterized as shown above. The ΦDESPT recovered with addition of F16Y mutation; however, the double ESPT emission quantum yield was slightly higher with Q108K:K40E:T53A:Q38F:Q4F:R58H:F16Y:A33C compared to the 146 Q108K:K40E:T53A:Q38F:Q4F:R58H:F16Y:A33M mutant (entries 6 and 7). The smallest ΦDESPT was obtained with Q108K:K40E:T53A:Q38F:Q4F:R58H:F16Y:M20Y (entry 9). The iminium pKa of this mutant is 5.9 resulting in residual PSB formation at neutral pH in the ground state. In addition, an interesting observation was made while measuring photophysical properties of this mutant: excitation of the PSB peak that appears as a shoulder at 520 nm leads to a low-intensity emission peak maximized at 674 nm, which is the wavelength for photoacidic single ESPT complexes. PSB-OH SB-OH PSB-O DESPT PSB-OH PSB-O SESPT Figure III-16. Absorption (left) and emission spectra (right) of Q108K:K40E:T53A:Q38F:Q4F:R58H:F16Y:M20Y/ThioPhenol complex. Upon excitation of SB (blue line) and excitation of the PSB (red line). DESPT= Double ESPT. SESPT= Single ESPT. Overlaying the emission spectra when exited at SB (380 nm) and PSB (520 nm) proves that the small shoulder in the right side of the double ESPT emission 147 of M4/ThioPhenol complex correlates to the photoacid single ESPT as discussed in Section III.5.1 (Figure III-16). III.6. Application of the double ESPT systems as a fluorescent tag for live cells imaging We chose to explore the performance of hCRBPII/ThioPhenol double ESPT complexes as a no-wash live-cell imaging tag. The mutant Q108K:K40E:T53A:Q38F:Q4F:R58H/ThioPhenol (M4) was chosen for these studies. This mutant shows a low iminium pKa leading to pure SB formation in the ground state. This complex yielded the highest ΦESPT and fluorescence quantum yield. Moreover, size exclusion chromatography data indicates that M3 is expressed solely in the monomeric form, which is advantageous for imaging purposes. All spectroscopic properties of the ThioPhenol/M4 complex are summarized below. Notably, the fast binding enables a quick labeling protocol with 5 min staining at 37 °C. Table III-7. Spectroscopic properties of ThioPhenol/M4 complex. e t1/2 b Complex λabs λem ΦESPT Φa pKa (M-1.cm-1) (min) ThioPhenol/M4 370 612 35,081 0.98 0.16 <5 <3 a Absolute quantum yield was measured on a Quantaurus-QY. b Half-life based on the rate constant obtained from Second order rate fitting; measured at 23 °C with 20 μM protein and 0.5 equiv ThioPhenol at pH 7.2. The M3-hCRBPII was cloned into the pFlag-CMV2 vector containing EGFP fused on the N-terminus of hCRBPII to label the whole cell. Additionally, to target 148 M4 at cell nuclei, the signaling peptide NLS (nuclear localization sequence) was fused to the C-terminus of M4 (see Figure II-32). Cells were transfected, and the fused constructs were expressed in HeLa cancer cell lines. Imaging was performed by incubating the HeLa cells with 10 μM ThioPhenol for five minutes at 37 °C. The cells were then directly subjected to confocal imaging without any washing steps prior to imaging. In all cases, the green fluorescence was observed upon excitation at 488 nm, indicating that transfection was successful, and the fusion protein was expressed (Figure III-17, the green channel). Stained cells were then excited via a 405 nm laser to collect the far-red emission in the 550 nm-650 nm window (Figure III-17, the red channel). Apparently, no fluorescence background is observed in the red channel, indicating that Thiophenol does not label off-target lysines or non-specific bindings does not lead to fluorescence signal. These results prove the utility of no-wash labeling of Q108K:K40E:T53A:Q38F:Q4F:R58H mutant with ThioPhenol in mammalian cells with no fluorescent background. 149 EGFP-whole cell ThioPhenol/hCRBPII-whole cell Ex: 488 nm Ex: 405 nm Em: BP 500 nm- 550 nm Em: BP 550 nm- 650 nm DIC Ex: 488 nm Green + Red channel BP 500 nm- 550 nm Figure III-17. Confocal imaging of labeled HeLa cells expressing EGFP-hCRBPII- whole cell. Cells were stained with 10 µM ThioPhenol and incubated at 37 °C for 5 min. Cells were not washed before imaging. 150 III.7 Enhancing the double ESPT process with brighter chromophore cores Attempts to enhance the quantum efficiency of the dual ESPT emission through the insertion of basic residues in the vicinity of the ThioPhenol’s hydroxyl group were not fruitful. The highest ΦDESPT and fluoresce quantum efficiency was acquired with the Q108K:K40E:T53A:Q38F:Q4F:R58H- (M4)/ThioPhenol complex, 98% and 16%, respectively, the very first complex we tested to examine the double ESPT process. Association of R58H with other proton acceptor residues leads to either the same photophysical characteristics as M4 or reduced ΦDESPT as the result of contribution from photobase single ESPT emission (see Tables III-4 and III-6). As discussed in Chapter II, free ThioPhenol is not emissive due to its weak ICT in the ground state. Deprotonation of the hydroxyl group, which occurs upon excitation produces the corresponding alkoxide, a strong electron-donating group, leading to fluorescence activation. This system is perfect for no-wash background- free imaging applications with unprecedented fluorescent signal specificity, as the deprotonation happens only upon binding to the engineered target protein. However, in comparison to other commercial fluorophores such as FRs, rhodamine, or cyanine dyes, the brightness of our system needs to be improved. Therefore, the next part of this chapter describes our attempts to increase the brightness of the double ESPT system while the specificity is kept intact. Hence, in the next step, we sought to investigate if the double ESPT process is possible with brighter chromophore cores such as FR0 and FR1. 151 III.7.1 MR0 To examine the possibility of the double ESPT process with other chromophores, we decided to optimize the structure of FR0. This scaffold was selected for its extremely high quantum yield and relatively high extinction coefficient. Mr. Mehdi Moemeni successfully synthesized and purified MR0, wherein the N,N diethyl group is substituted with the hydroxyl group (Figure III-18, see Section IV-7 for detailed synthesis). H H H S HO NN O O O HO O ThioPhenol MR0 FR0 FR0 Figure III-18. Structures of ThioPhenol, MR0, and FR0. III.7.1.A. Spectroscopic properties of MR0 in solution First, we measured MR0 absorption, emission, and quantum yield in various organic solvents with different polarities to investigate its spectroscopic behavior. Results are summarized in Tables III-7. MR0 shows almost the same absorbance wavelength in different solvents, with the maximum centered around 339 nm. On the other hand, the maximum emission wavelength is more sensitive to the environment’s polarity; spanning from 382 nm in toluene to 568 nm in aqueous solvents (see Figure III-19 and Table III-7). 152 The most interesting result of this study is, however, the significant increase of the fluorescence quantum yield upon increasing solvent polarity. MR0 is not emissive in less polar solvents; the quantum yield is less than 0.01 in toluene and ethyl acetate, but it reaches to 0.33 in ethanol. Fortunately, these characteristics help to eliminate the fluorescent background from unbound MR0 in the hydrophobic cell environments. Table III-8. Spectroscopic characterization of MR0 in various solvents. ε Entry Solvent λabs (nm) λem (nm) SS (nm) Φa (M-1cm-1) 1 Toluene 341 382 41 30,025 0.00 2 Ethyl acetate 338 394 56 29,548 0.00 3 Dimethyl sulfoxide 334 434 90 30,501 0.13 4 Ethanol 341 459 118 23,511 0.33 5 PBS buffer 339 492/568 153/229 25,974 0.15 a Absolute quantum yield was measured on a Quantaurus-QY. Next, we compared the spectral properties of the three chromophores, FR0, MR0, and ThioPhenol, in non-polar (toluene), and polar (ethanol) environments (Table III-8). FR0 shows the highest quantum yields, while ThioPhenol is not emissive as a free aldehyde regardless of the solvent polarity. However, MR0 exhibits zero quantum yield in non-polar solvents and becomes bright in ethanol. 153 Although there is the same number of conjugated double bonds in MR0 and FR0 structures, the absorption wavelength of the former is blue-shifted by 55 nm due to the less electron-donating ability of the hydroxyl group. MR0 absorption wavelength is blue-shifted by about 50 nm compared to ThioPhenol as well. Nonetheless, the brightness (e * Φ) of MR0 is 26 times higher than ThioPhenol. Table III-9. Spectroscopic features of MR0, FR0, and ThioPhenol in toluene and ethanol. MR0 FR0 ThioPhenol λabs λem Φa λabs λem Φa λabs λem Φs Toluene 341 381 0.00 396 434 0.70 386 461 0.0 Ethanol 341 459 0.33 396 556 0.66 391 515 0.01 a Absolute quantum yield was measured on a Quantaurus-QY. a. b. Figure III-19. Spectroscopic properties of MR0 in different solvents. a. UV-Vis and b. Fluorescence spectra. 154 An interesting observation made in this study is that free MR0 emission measurement in water or other aqueous solutions such as BPS buffer results in two maxima at 492 nm and 568 nm, respectively (see Figure III-19 and Table III- 7). Hence, we speculated that the red-shifted second peak at 568 nm might be due to the deprotonation of the MR0 hydroxyl group. To confirm if this assumption is correct, the absorption and emission of MR0 were measured upon acid and base titrations (Figure III-20). a. b. Figure III-20. UV-Vis spectra (left) and fluorescence spectra (right) of free aldehyde MR0 upon acidification and basification of the sample. Measurements were done in water. The absorption and emission spectra of MR0 did not change upon acidification of the sample to pH 2.5. However, increasing the pH of the sample to 10 red shifts the absorption wavelength from 338 nm to 384 nm (46 nm). Apparently, deprotonation of the hydroxyl group of MR0 results in the red-shifted absorption. 155 Interestingly, excitation of the MR0-phenolate leads to the emission wavelength maximized at 577 nm, close to the red-shifted emission peak of MR0. This observation verifies that in contrast to ThioPhenol, about half of the MR0 becomes deprotonated in aqueous solutions at neutral pH (results are summarized in Table III-9). Table III-10. Spectroscopic features of free aldehyde MR0 in water in acidic and basic pH. Free Aldehyde MR0 λabs λem SS (nm) Neutral/Acidic pH 338 492/570 154/232 Basic pH 384 578 194 Next, in order to mimic the product of the protein bound complex, the aldehyde was coupled with n-butyl amine in ethanol (Figure III-21). As was previously discussed for ICT systems, it is expected that the reduced electron- withdrawing ability of the acceptor results in a blue-shift of the absorption and emission wavelength. Accordingly, in this system, imine formation leads to 9 and 54 nm blue-shifts in the absorption and emission wavelength, respectively, due to the replacement of the oxygen atom with the less electronegative nitrogen atom. Furthermore, to better characterize this system before moving into the complex environment of the protein cavity, the imine sample was acidified and basified, and the photophysical properties of PSB and phenolate-SB-complex were measured (the results are summarized in Table III-9). 156 a. HO N H Acid Phenol-PSB H HO HO O n-Butylamine N MR0 EtOH Phenol-SB Base O N b. Phenolate-PSB PSB-OH PSB-OH SB-OH SB-O PSB-O Figure III-21. a. Schiff base and protonated Schiff base (PSB) of MR0 with n-butyl amine in ethanol. b. Absorbance (left) and emission (right) spectra of MR0 and derivatives: Phenol-SB, Phenol-PSB, and Phenolate-PSB. Most of the MR0 forms Phenol-SB upon condensation with n-butylamine; however, a small portion of the product is protonated, which appears as a small shoulder to the right side of the SB absorption peak at about 383 nm. Excitation of Phenol-SB complex at its maximum absorption wavelength (332 nm) leads to an emission spectrum with maxima at 405 and 509 nm. Apparently, the phenol-SB complex acts as a photobase and can abstract a proton upon photoirradiation, 157 resulting in a Stokes shift of more than 175 nm. The maximum emission wavelength of the Phenol-PSB complex at 510 nm verifies the accuracy of this statement. Table III-11. Spectroscopic characterization of MR0 and derivatives. ε Compound λabs λem SS (nm) Φa ΦESPT (M-1.cm-1) Free Aldehyde MR0 341 459 118 23,511 0.33 - Phenol-SB 332 405/509 73/177 16,928 0.11 51.2 Phenol-PSB 398 510 112 12,360 0.40 - Phenolate-SB 375 489/628 114/253 18,406 0.07 48.6 a Absolute quantum yield was measured on a Quantaurus-QY. Interestingly, excitation of the Phenolate-SB produces a two-maxima emission peak as well at 489 and 628 nm, corresponding to Phenolate-SB and Phenolate-PSB complexes, respectively. This highlights the strong photobasic characteristics of the MR0-SB complex; even in such a basic environment where even though the hydroxyl group is deprotonated, photoexcitation of SB leads to PSB formation. Such photobasic properties were not observed for ThioPhenol in solution (see Section II. 5). Nonetheless, MR0 photoacidic properties are not as strong, since photoirradiation of the Phenol-PSB complex shows a small peak at 650 nm, which might be due to the deprotonation of the hydroxyl group (Figure III- 21b). 158 To investigate further about photobasic features of MR0, we chose to measure the absorption and emission of the phenol-SB in PBS, which might better resemble the protein cavity environment (Table III-10). Both the absorption and emission wavelength of Phenol-SB show bathochromic shifts, and the ΦESPT (the percentage of conversion to PSB upon excitation) is significantly increased (>90%) in BPS buffer as compared to when measured in ethanol. Table III-12. Spectroscopic properties of MR0 Phenol-SB in PBS buffer. ε Compound λabs λem SS (nm) ΦESPT (M-1.cm-1) Phenol-SB 348 470/598 122/250 18,439 92.6 Same as FR0, the strong photobasic activity of MR0 could be detrimental for developing selective low-background fluorescent tags. However, this section aims to investigate whether the double ESPT process is possible with brighter chromophore structures, which then facilitates studying the kinetics of this unique phenomenon. Nonetheless, the emission of the double ESPT process is most red- shifted and is well-separated from the Phenol-PSB emission; thus, the chances of background form cross-talk emissions will be diminished. III.7.1.B. Attempts to develop double ESPT process with MR0-hCRBPII complex As discussed previously, FR1, ThioFluor, and ThioPhenol exhibit photobasic properties in complexation with M3. Additionally, M4 is the optimized 159 mutant for the double ESPT process. Hence, we decided to measure the photophysical properties of MR0 with Q108K:K40E:T53A:R58L:Q38F:Q4F (M3) and Q108K:K40E:T53A:R58H:Q38F:Q4F (M4) hCRBPII mutants. Evident from the absorption, MR0 is bound as a SB to both mutants. According to in-solution studies with MR0, the photogenerated Phenol-PSB emission wavelength is about 510 nm, which correlates with the M3 complex emission wavelength (Table III-11, entry 1). a Absolute quantum yield was measured on a Quantaurus-QY. Interestingly, the emission wavelength with M4 (the double ESPT mutant) Table III-13. Spectroscopic properties of MR0/M3 and MR0/M4. Entry Complex λabs λem SS Φa 1 MR0/M3 348 505 157 0.56 2 MR0/M4 347 525 178 0.31 is only 20 nm more red-shitted than M3. We expected that the double ESPT derived emission to be more red-shifted as the photogenerated Phenolate-PSB complex maximum emission wavelength is 628 nm in ethanol. Furthermore, the fluorescence quantum efficiency of MR0/M3 is higher, which might be due to the absence of the double ESPT process with the MR0/M4 complex. As discussed in Section III.5.1, in contrast to Phenolate-PSB, Phenol-PSB or Phenolate-SB complexes are unable to produce a strong ICT system and are less emissive (see Table III-3). 160 We presumed that the photoexcitation of the MR0/M4 complex might lead to deprotonation of the hydroxyl group and Phenolate-SB complex formation. To test this hypothesis, we measured the photophysical properties of the MR0/M4 and MR0/M3 complexes in basic pH (Table III-12). In addition, we chose to explore and compare the photophysical properties of MR0 with Q108K:K40L:T51V:T53S:R58H:F16Y:A33H:L117C (M5) mutant that shows photoacidic characteristics with ThioPhenol (Table III-12). The absorption and emission spectra of MR0 with M3, M4, and M5 mutants were monitored upon base titration of their solutions in PBS buffer (Figure III-22). Table III-14. Spectroscopic properties of MR0/M3 and MR0/M4, and MR0/M5. hCRBPII Neutral pH 7.3 Basic pH 10 Entry Mutant a λabs λem λabs λem 1 M3 348 505 336/371 430/576 2 M4 347 525 332/364 430/575 3 M5 340 493/572 382 576 a M3: Q108K:K40E:T53A:R58L:Q38F:Q4F (photobase mutant), M4: Q108K:K40E:T53A:R58H:Q38F:Q4F (double ESPT mutant), M5: Q108K:K40L:T51V:T53S:R58H:F16Y:A33H:L117C (photoacid mutant). Interestingly, all mutants show a two-maximized emission peak upon basification of the sample. The maximum emission wavelengths are the same for all mutants; however, the ratio of the red-shifted peak at 575 nm to the blue-shifted one at 430 nm is different (see Figure III-22, emission spectrum at pH ~10, the green line). Nonetheless, monitoring the UV-Vis spectra of these mutants upon 161 basification reveals that the absorption spectrum measured at pH ~10 (the green line) shows an irregular shape, and, in fact, it consists of two peaks. a. b. c. Figure III-22. Absorption (left) and emission (right) spectra of M3 (a), M4 (b), and M5 (c) upon basification of their solutions. 162 Upon comparing each mutant's absorption and emission spectra at basic pH, we realized that the blue and red-shifted peaks in absorption and emission are in relation to each other. For example, for the M3 complex, the absorption peaks at 336 and 371 nm have the same intensity, resulting in the broad absorption peak. Accordingly, the emission spectrum of M3 shows two equal emission peaks at 430 and 576 nm (Figure III-22a). On the other hand, the blue-shifted absorption has a higher intensity for the M4 complex, resulting in higher intensity for emission peak at 430 nm (Figure III-22b). H OH HO Active Site HO O O N NaOH. 1M N H Lys108 MR0 Phenol-SB Hydrolyzed Phenolate-SB λabs 339nm λabs 334nm λem 492 nm λem 430 nms Figure III-23. Formation of the hydrolyzed Phenolate-SB complex produced upon basification of the protein solution. Prior solution studies showed that none of the MR0 species absorb as blue- shifted as 336 nm. Additionally, emission at 430 and 575 nm at a high pH could not match any bound MR0 derivatives. We speculated that the basification of the sample with aqueous sodium hydroxide (1M. NaOH) solution leads to the hydrolyzation of the imine bond. At the same time, in the basic environment at pH ~10, the hydroxyl group gets deprotonated. Thus, the blue-shifted absorption and emission peaks with maximum wavelength at about 330 and 430 nm correspond to the phenolate hydrolyzed Phenolate-SB complex (see Figure III-23). Precise 163 characterization of the products is required to verify this statement. Presumably, the red-shifted absorption and emission peak at about 370 and 576 nm corresponds to the deprotonated hydroxyl group of non-hydrolyzed imines, Phenolate-SB complex (Figure III-23). As discussed above, the reason we were interested in the photophysical properties of the MR0/M5 complex is that this protein mutant, Q108K:K40L:T51V:T53S:R58H:F16Y:A33H:L117C, is engineered to show a high iminium pKa with ThioPhenol (8.8). However, MR0 binds this mutant as pure SB with low iminium pKa (<5). We sought to investigate whether MR0 can bind hCRBPII as an iminium; hence, it was incubated with Q108K:K40L:T51V:T53S:R58H:F16Y:Y19W:A33H:L117E mutant, which exhibit high pKa with both ThioPhenol and ThioFluor due to the L117E mutation. a. b. 338 496 335 490 565 Figure III-24. Absorption (blue line) and emission (red line) of Q108K:K40L:T51V:T53S:R58H:F16Y:Y19W:A33H:L117E/MR0 complex at neutral (left) and acidic (right) pH. 164 We measured the absorption and emission spectra of Q108K:K40L:T51V:T53S:R58H:F16Y:Y19W:A33H:L117E/MR0 complex in both neutral and acidic pH (Figure III-24). Surprisingly, as shown, even acidification of the solution to pH less than five did not lead to MR0 iminium formation. However, the red-shifted shoulder seems to be the PSB emission produced upon photoirradiation of SB (Figure III-24b). In general, it seems MR0 cannot bind hCRBPII as an iminium, but it can generate PSB upon photoexcitation. As described earlier, basification of the protein solutions showed that the MR0 SB-Phenolate complex emits around 575 nm, but it does not help much to characterize the MR0/M3 and M4 complexes emissions at 505 nm and 525 nm, respectively. Presumably, the collected emission spectrum is not from a single species, and the broad shape of the peak is an indication of other species formed upon excited-state proton transfer to the imine or from the hydroxyl group (see Figure III-22). Nonetheless, it is clear that the emission of MR0/M4 complex is not due to the double ESPT process, as solution studies suggested that the MR0 Phenolate-PSB complex would emit beyond 600 nm. In the next step, we sought to explore whether the introduction of proton- acceptor residues around the MR0 hydroxyl group would facilitate the double ESPT process. Unfortunately, there are no crystal structures from MR0 bound to hCRBPII. Thus, the most effective mutations in increasing the ΦESPT with ThioPhenol such as F16Y, T29Y, A33H, A33M, and L77M were introduced to M4, 165 the mutants yielded the highest ΦESPT, in hopes of improving the double ESPT process (Table III-14). Table III-15. Spectroscopic properties change as the result of the addition of proton acceptor residues. Entry hCRBPII Mutant a λabs λem SS (nm) ΦDESPT 1 KEAFF:R58H 347 525 178 0.02 > 2 KEAFF:R58H:T29Y 348 516 168 0.08 3 KEAFF:R58H:L77M 341 525 184 0.11 4 KEAFF:R58H:F16Y 329 560 231 0.53 5 KEAFF:R58H:A33H 334 512 178 0.07 6 KEAFF:R58H:F16Y:A33H 326 567 239 0.58 7 KEAFF:R58H:Y19W:A33H 345 498 159 0.43 8 KEAFF:R58H:F16Y:T29Y 329 562 233 0.57 9 KEAFF:R58H:F16Y:L77M 330 493/562 163/232 0.54 10 KEAFF:R58H:F16Y:A33M 329 435/565 106/236 0.61 a KEAFF equals to Q108K:K40E:T53A:Q38F:Q4F. As listed above, the largest Stokes shifts (>230 nm) were acquired with mutants containing F16Y, entries 4 and 6, leading to the maximum emission wavelength around 565 nm. The addition of other mutations such as A33H, T29Y, and L77M either did not change or blue-shifted the emission wavelength compared to the M4 complex (entry 1). The Phenolate-PSB complex produced upon single ESPT of imine to iminium in ethanol emits around 630 nm with Stokes shift more than 250 nm (Table III-10). However, none of the tested protein mutants could result in such red-shifted emission. As was observed with ThioPhenol, the emission of Phenolate-PSB is 166 always blue-shifted for the double ESPT process as compared to its formation upon a single ESPT process (Figure III-25). O O N Imine Single ESPT N H In EtOH Phenolate-SB Phenolate-PSB λabs 375 nm λem 628 nm λem 489 nm HO O N Doubel ESPT N H Phenol-SB Phenolate-PSB λabs 330 nm λem 567 nm H H N Hydroxyl group N S Single ESPT S HO O ThioPhenol-PSB ThioPhenolate-PSB λabs 520 nm λem 680 nm λem 575 nm H N N Doubel ESPT S HO S O ThioPhenol-SB ThioPhenolate-PSB λabs 370 nm λem 612 nm Figure III-25. The schematic representation of single and double ESPT processes and their photophysical properties. 167 The K40E mutation is retained in all double ESPT mutants as the proton source, and thus could putatively lead to the localization of the iminium positive charge and its blue-shifts in emission. Therefore, we surmised that the emission around 570 nm for entries 4 and 6 could be from the double ESPT process. In contrast to double ESPT systems with ThioPhenol that can provide quite a narrow emission, all mutants tested with MR0 produced a broad emission spectrum (Figure III-26). We attempted to narrow the emission spectrum by adding other proton acceptor residues besides F16Y; however, this was not fruitful (Table III-6, entries 8-10). 330 nm 560 nm 497 nm Figure III-26. Absorption and emission spectra of Q108K:K40E:T53A:Q38F:Q4F:R58H:F16Y/MR0 complex. The blue-shifted shoulder corresponds to Phenol-PSB, or Phenolate-SB produced upon single ESPT, and the emission at 560 nm results from Phenolate-PSB complex produced upon the double ESPT process. 168 As discussed at the beginning of this section, one of our goals is to develop double ESPT systems with higher fluorescent quantum yield. MR0 is a bright chromophore that binds designed hCRBPII mutants as a SB within 1 or 2 minutes. However, MR0 complexes are not selective; photoirradiation of the SB results in other ESPT products besides Phenolate-PSB (the product of double ESPT), leading to a broad emission band. For instance, as shown in Figure III-26, excitation of Phenol-SB at 330 nm leads to a broad emission spectrum. Presumably, the left shoulder maximized at 497 nm results from imine to iminium or phenol to phenoxide single ESPT, and the peak at 560 nm is correlated to the Phenolate-PSB complex. More experiments are required to be able to characterize the emission precisely. On the other hand, the live-cell applications are limited because, as discussed in Section III.7.1.B the MR0 complexes are unstable and tend to hydrolyze easily in basic or acidic environments. Additionally, the absorption and emission wavelengths with MR0 are blue-shifted, and the double ESPT Stokes shift is smaller as compared to ThioPhenol. As shown in Table III-6, all MR0 SB complexes absorb in the UV region of the spectrum (less than 350 nm), which again can hamper its utility. III.7.2 MR1 Next, we continued to pursue our goal through optimization of the FR1 structure. Mr. Mehdi Moemeni successfully synthesized MR1 wherein N,N diethyl nitrogen is replaced with a hydroxyl group (Figure III-27). FR1, a derivative of FR0, 169 was first designed and synthesized by Dr. Wei Sheng and exhibited the optimal extinction coefficient, fluorescence quantum yield, and proper absorption and emission wavelength. N HO O O FR1 MR1 Figure III-27. Structures of FR1 and MR1. Dr. Wei Sheng successfully reported the first photoswitchable NIR tags that employ a synthetic dye, FR1, and a fusion protein. FR1/hCRBPII complexes showed great ability to photoactivably turn “ON” and turn “OFF” the fluorescence signal using an ICT-capable chromophore.44 III.7.2.A. Spectroscopic properties of MR1 in solution To know about the spectroscopic characteristics of the newly designed chromophore, we measured its photophysical properties in various organic solvents with different polarities (Table III-15). Table III-16. Spectroscopic characterization of MR1 in various solvents. ε Entry Solvent λabs (nm) λem (nm) SS (nm) Φa (M-1cm-1) 1 Toluene 364 412 48 83,890 0.03 2 Ethyl acetate 357 398 41 86,611 0.04 3 Dimethyl sulfoxide 369 497 128 71,162 0.30 4 Ethanol 368 525 157 62,437 0.23 5 PBS buffer 362 562 200 51,230 0.03 a Absolute quantum yield was measured on a Quantaurus-QY. 170 As shown above, in Table III-15, the absorption wavelengths are close in different solvents centered around 364 nm. However, the emission wavelength shifts bathochromically in more polar solvents; it shifts more than 160 nm, from 398 nm in toluene to 562 nm in PBS buffer. FR1 shows a strong solvatochromism in its fluorescence in various solvents (see Figure III-28 and Table III-15). Such emission solvatochromism was expected since, like FR1, the polar excited state of MR1 is more prone to solvent relaxations. a. b. Figure III-28. Spectroscopic properties of MR1 in different solvents. a. UV-Vis and b. Fluorescence spectra. Interestingly, the fluorescence quantum efficiency of MR1 in less polar solvents such as toluene and ethyl acetate is less than 5% (Table III-15, entries 1 and 2). Moreover, MR1 is not quite soluble in aqueous solutions such as PBS buffer and shows a quantum yield of 3% (entry 5). These properties are advantageous for imaging applications as the free unbound MR1 is not emissive in hydrophobic or aqueous parts of the cell milieu and thus, eliminates the need to 171 wash off free chromophore prior to imaging. Furthermore, in contrast to FR1, no deprotonation of the hydroxyl group was observed upon excitation in BPS buffer, leading to a less fluorescent background for fluorescent confocal imaging; since the corresponding alkoxide emission is red-shifted and might leak to the emission window of the designed fluorescent complex. Comparing the photophysical properties of ThioPhenol with MR1 in the same solvents indicates that for the latter, the average absorption and emission wavelengths have red-shifted 23 nm and 65 nm, respectively. In addition, the extinction coefficient and fluorescence quantum yield of MR1 are significantly higher and lead to a 48-fold increase in the total brightness of the chromophore (Table III-16). Table III-17. Comparison of spectroscopic features of free aldehyde ThioPhenol and MR1 in ethanol. ε Brightness Chromophore λabs λem SS (nm) Φ (M-1.cm-1) (e*Φ) ThioPhenol 342 462 120 29,752 0.01 298 MR1 368 525 157 62,437 0.23 14,361 a Absolute quantum yield was measured on a Quantaurus-QY. As for previous chromophores, it is essential to characterize the newly designed dye in solution before moving to the complex environment of the protein’s 172 cavity. Hence, to mimic the product of MR1 condensation in the protein pocket, the aldehyde was coupled with the n-butyl amine in ethanol (Figure III-29). a. HO n-Butylamine HO O N MR1 Phenol-SB Base HO Acid O N N H Phenol-PSB Phenolate-SB b. Figure III-29. a. Schiff base and protonated Schiff base (PSB) of MR1 with n- butylamine in ethanol. b. Absorbance (left) and emission (right) spectra of MR1 and derivatives: Phenol-SB, Phenol-PSB, and Phenolate-PSB. As described at the beginning of this chapter (Section III-1), FR0 possesses superior photobase activity. Its derivative MR0 is a strong photobase as well that 173 even in a basic solution where the hydroxyl group is deprotonated, the corresponding imine can abstract a proton from ethanol the (see Figure III-21 and Table III-10). It is clear from solution studies that FR1 and MR1 are weaker photoacids/bases. Excitation of the Phenol-SB activates the imine as a photobase and results in a two-maxima emission spectrum at 445 nm, and 564 nm, which corresponds to Phenol-SB and PSB, respectively (Table III-17, second entry). The emission of Phenol-PSB produce in the ground state upon acidification of the solution at 575 nm can verify this claim. However, in contrast to MR0, no imine ESPT was observed for Phenolate-SB (third and fourth entry). Table III-18. Spectroscopic characterization of MR1 and derivatives. ε Compound λabs λem SS (nm) Φa ΦESPT (M-1.cm-1) Free Aldehyde MR1 368 525 157 62,437 0.33 - Phenol-SB 355 445/564 90/209 71,727 0.16 43.9 Phenol-PSB 427 575 151 83,544 0.13 - Phenolate-SB 401 541 140 70,117 0.04 <2 a Absolute quantum yield was measured on a Quantaurus-QY. III.7.2.B. Exploration about photoacidic and photobasic properties of MR1 complexes In the first step, we sought to investigate whether MR1 can act as a photobase in the protein environment. Thus, we measured its photophysical properties upon binding with the M3 mutant, which is optimized as the best photobase in complexation with ThioFluor and FR1 chromophores (Table III-18). 174 Monitoring the UV-Vis spectra indicates that the binding is fast, as the MR1 Phenol-SB complex forms within 1-2 minutes. In addition, the imine pKa is less than 5 (slightly lower than the previous chromophore), resulting in pure SB formation in neutral pH and a narrow Phenol-PSB emission (Figure III-30). Nonetheless, the fluorescence quantum yield with MR1 has decreased to 26% due to the weak electron-donating effects of the hydroxyl group and failure to form an ICT system upon photoexcitation. SB-OH PSB-OH Figure III-30. The normalized absorption (blue line) and emission (red line) spectra of MR1/M3 complex. Table III-19. Comparison of the spectroscopic properties of FR1/M3 and MR1/M3 complexes. SS Entry Complex λabs λem Φa (nm) 1 FR1/M3 b 392 595 203 0.72 2 MR1/M3 357 502 145 0.26 a Absolute quantum yield was measured on a Quantaurus-QY. b M3: Q108K:K40E:T53A:Q38F:Q4F:R58L. 175 As shown in Section III.7.1.B, MR0 could not form PSB with hCRBPII mutants; even with mutants containing L117E, the iminium pKa was less than 5. Therefore, in order to study the MR1 iminium and explore if it can show photoacidic properties the same as ThioPhenol, we chose to measure the photophysical characteristic of MR1 and Q108K:K40L:T51V:T53S:R58H, the mutant that showed ΦESPT of 51% with ThioPhenol. Additionally, as a control experiment, MR1 was incubated with Q108K:K40L:T51V:T53S mutant, which lacks the essential mutation (R58H) for deprotonation of the hydroxyl group (Table III-19). The imine pKa is low with both mutants resulting in a small amount of minimum formation in the ground state. The Q108K:K40L:T51V:T53S:R58H/MR1 complex resulted in a rather broad emission spectrum with a maximum at 461 nm, which most probably is due to the Phenol-SB emission. On the other hand, unexpectedly, the Q108K:K40L:T51V:T53S/MR1 complex behaved as a photobase since its emission wavelength is the same as M3/MR1 complex with Stokes shift of more than 200 nm (Table III-19, entry 1). Table III-20. Spectroscopic changes as the result of addition of R58H mutation. Entry hCRBPII mutant λabs λem SS (nm) 1 Q108K:K40L:T51V:T53S 348 501 203 2 Q108K:K40L:T51V:T53S:R58H 345 461 145 This phenomenon was observed with ThioPhenol previously as well. As described in detail in Section III-3 and Table III-2, ThioPhenol can appear as a 176 photobase even when K40E mutation is substituted with K40L in M3 mutant. Nonetheless, the Q108K:K40L:T51V:T53S:R58H/MR complex was acidified to investigate if the PSB can act as a photoacid. Interestingly, excitation at the PSB absorption wavelength (500 nm) results in a two-maxima emission spectrum at 602 nm and 692 nm. The latter red-shifted emission corresponds to the Phenolate-PSB complex product of the photoacid process formed upon ESPT of the hydroxyl group (Figure III-31). a. b. Ex at 500nm 345nm 461nm 692nm 602nm 345nm Figure III-31. The absorption and emission spectra of Q108K:K40L:T51V:T53S:R58H/MR1 complex a. upon SB excitation in neutral pH 7.2 and b. PSB excitation in acidic pH 5.4. Notably, same as FR1 complexes, Q108K:K40L:T51V:T53S:R58H/MR1 represents photoswitchable properties. Upon binding, the complex shows a maximum absorption wavelength at 345 nm with a minor peak at 600 nm. A 30- second UV irradiation (using a ~365 nm handheld UV lamp or a Xenon lamp 177 equipped with BP 300-400 band filter) can switch the thermal “OFF” state to a kinetic “ON” state that has a predominant absorption at 500 nm (Figure III-32). Excitation of the “OFF” state at 345 nm results in an intense 461-nm blue emission, with a tail around 600 to 680 nm. “ON” state excitation at 500 nm results in the same emission obtained upon acidification of the solution (see Figure III-31). OFF-state, predominant state trans-SB prior to UV irradiation N N UV light HO HO λabs 345nm λem 461nm low pKa high pKa predominant state ON-state, post UV irradiation cis-PSB N Visible NH H HO HO light λabs 500nm λem 692nm Figure III-32. Proposed photoswitching cycle of Q108K:K40L:T51V:T53S:R58H/MR1 complex. Studies of the photoswitchable properties of MR1 were not pursued any further as this section aims to investigate whether it is possible to develop the double ESPT process with MR1 complexes. Besides, Dr. Wei Sheng has studied and described this phenomenon with FR1 chromophore previously. 44 Experiments with Q108K:K40L:T51V:T53S:R58H/MR1 complex indicates that MR1 Phenol-PSB can show photoacidic properties; however, only in an acidic 178 solution, where a decent amount of PSB is present. We attempted to explore if MR1 can appear as a photoacid in neutral pH or form stable PSB in the ground state. To this end, two protein mutants that show relatively high ΦESPT and iminium pKa with ThioPhenol were chosen to test with MR1 (Table III-20). Table III-21. Spectroscopic properties of MR1 with high iminium and mutants Entry hCRBPII mutant a λabs λem SS (nm) pKa 1 KLVS:R58H:A33H:F16Y:L117C 363 519 156 5.2 2 KLVS:R58H:Y19W:A33H:F16Y:T29Y:L117C 368 550 182 <5 a KLVS is equal to Q108K:K40L:T51V:T53S. As listed above, binding to both mutants gives low iminium pKa, and as a result, Phenol-SB is the dominant formed species in neutral pH. There is about a 10 nm red-shift in the SB absorption wavelength compared to the MR1/M3 complex. Nonetheless, excitation of the SB in Q108K:K40L:T51V:T53S:R58H:Y19W:A33H:F16Y:T29Y:L117C/MR1 complex gives a broad emission maximized at 550 nm, which is too red-shifted to be SB emission and thus, presumably is the emission of SB-Phenolate complex. However, the emission spectrum is broad enough for both mutants suggesting other species besides the Phenolate-SB. To confirm if the hydroxyl group becomes deprotonated upon excitation of the Phenol-SB, we decided to monitor the absorption and emission spectra of the same complex upon acid and base titration of its solution (results are summarized in Table III-21). 179 The absorption and emission wavelengths of Q108K:K40L:T51V:T53S:R58H:Y19W:A33H:F16Y:T29Y:L117C/MR1 in acidic and basic pH are listed below. Due to the low iminium pKa, acidic pH (5.1) a tiny fraction of the PSB peak was observed at 465 nm, which its excitation leads to ESPT of the hydroxyl group and Phenolate-PSB formation. Basifying the protein solution to pH more than 10 deprotonates the hydroxyl group in the ground state and gives the Phenolate-SB complex. Interestingly, the emission of this complex at 548 nm is close to the emission wavelength in neutral pH, which verifies that MR1 complexes can appear as a strong photoacid however the iminium pKa should increase. Table III-22. Spectroscopic properties of MR1 with high iminium and mutants. Acidic pH 5.1 Neutral pH 7.3 Basic pH 10.7 λabs λem SS λabs λem SS λabs λem SS 465 571/677 106/212 368 550 182 410 548 138 In the next step, we sought to investigate whether it is possible to enhance the photoacidic properties of MR1 complexes, whether through increasing the iminium pKa or inserting more proton acceptor residues around the hydroxyl group. MR1 is a much brighter dye than ThioPhenol, which emits in the far-red NIR region of the spectrum and can find deep tissue fluorescent imaging applications. To identify the closest residues to the hydroxyl group of MR1, we docked the energy-optimized MR1 structure into the crystal structure of 180 Q108K:K40E:T53A:R58W:Q38F:Q4F:Y19W/ThioFlour complex (Figure III-33). The docking simulation was done using the pair fitting function in Pymol, and it indicates that Q38F and L77 (if the MR1 structure is flipped) are the closest to the MR1 hydroxyl group (Figure III-33). The protein mutants obtained upon mutation of these residues and their properties are listed in Table III-22. L77 Q108K Q38F Figure III-33. Flexible docking of MR1 in the crystal structure of Q108K:K40L:T51V:T53S:R58W-hCRBPII/ThioFluor. MR1 is shown in purple, and ThioFluor is shown in cyan. As described in Chapter II in Sections II.11.1C and II.11.3A, tyrosine works best at positions 77 and 38 for protein expression yield and deprotonation of the hydroxyl group of bound ThioPhenol. Hence, we sought to investigate the effect of these mutations on the photoacidic properties of MR1. Comparing the emission wavelengths of entries 1 and 2 indicates that Q38Y mutation can effectively activate the photoacidity of the bound MR1. With this mutation, the emission 181 wavelength is at 500 nm, which corresponds to the Phenolate-SB complex; however, the emission wavelength with L77Y at 471 nm represents the Phenol-SB complex. Nonetheless, both mutants show a low iminium pKa, and no PSB formation is observed in neutral pH. Table III-23. Spectroscopic change as the result of L77Y and Q38Y mutations. high iminium and mutants Entry hCRBPII mutant a λabs λem SS (nm) 1 KLVS:R58H:A33H:Q38Y 350 500 150 2 KLVS:R58H:F16Y:L77Y 345 471 126 3 KLVS:R58H:Y19W:A33H:F16Y: L117C:L77Y 364 493/595 129/231 a KLVS is equal to Q108K:K40L:T51V:T53S. In studies with ThioPhenol, we realized that L117C mutation usually increases the iminium pKa, but notably, for all the same protein mutants, MR1 complexes show lower iminium pKa than ThioPhenol. The iminium pKa in Q108K:K40L:T51V:T53S:R58H:Y19W:A33H:F16Y: L117C:L77Y/MR1 is 5.4 and gives a slightly higher amount of PSB in neutral pH. In order to better characterize this system, the emission spectrum of this complex was collected upon excitation of the SB and PSB (Figure III-34). An interesting observation was made during this study; excitation of Phenol-SB results in a broad two-maxima emission spectrum at 493 nm and 595 nm. As discussed above with other protein mutants, the blue-shifted emission results from the hydroxyl group deprotonation in the excited state. The more intense hypsochromic shifted emission is presumably due to the double ESPT process that leads to more than 230 nm Stokes shift, the 182 largest Stokes shift of MR1/hCRBPII complexes up to this point. Surprisingly, this mutant is not equipped for proton transfer to imine (K40E); however, such a large Stokes shift verifies the occurrence of the double ESPT process. Furthermore, we surmised that the collected emission acquired upon excitation of the PSB-Phenol complex at 470 corresponds to the Phenolate-PSB complex (Figure III-34a). However, as discussed earlier, the emission of the Phenolate-PSB complex is more red-shifted when it is the product of the single ESPT process than the product of double ESPT, 661 nm, and 595 nm, respectively, for the current system. a. b. PSB-O Ex at 345nm 661nm PSB-O 595nm SB-O 493nm Ex at 470 Figure III-34. Absorption (blue line) and emission (red line) spectra of Q108K:K40L:T51V:T53S:R58H:Y19W:A33H:F16Y: L117C:L77Y/MR1 complex upon excitation of a. SB at 364 nm and b. PSB at 470 nm in neutral pH. We monitored the absorption and emission spectra of this complex over acid and base titrations as well. When measured in a basic solution (the dark blue line) with the neutral solution, the comparison of the Phenol-SB emission confirms that 183 the emission peak maximized at 493 nm is correlated to the deprotonated hydroxyl. The emission spectrum measured in the acidic pH (4.8 the purple line) is quite broad and could indicate the exitance of more than one species. Presumably, all Phenol-PSB, Phenolate-SB, and Phenolate-PSB complexes contribute to this emission (Figure III-35). a. b. Ex at 345nm Figure III-35. Absorption (left) and emission (right) spectra of Q108K:K40L:T51V:T53S:R58H:Y19W:A33H:F16Y: L117C:L77Y/MR1 upon acid and base titrations. Up to this point, all mutants which showed high iminium pKa with ThioPhenol led to a residual PSB formation with MR1 in neutral pH. However, previously, Dr. Wei Sheng demonstrated that FR1 binds Q108K:K40H:T53A:R58L:Q38F:Q4F (M7) mutant mainly as an iminium.45 Hence, we sought to investigate the photophysical properties of the MR1/M7 complex and explore if the excitation of the PSB can result in the deprotonation of the hydroxyl group and photoacidity. The iminium pKa of this complex is 6.9, which is the highest 184 pKa measured for MR1 complexes, leading to almost equal SB and PSB formation. The comparison of MR1/M7 absorption and emission wavelengths with FR1 is shown in Table III-23. Table III-24. Comparison of the spectroscopic properties of FR1/M7 and MR1/M7 complexes. SB SB SS PSB PSB SS Entry Complex pKa λabs λem (nm) λabs λem (nm) 1 FR1/M7a 398 450/630 52/232 516 630 114 >7 2 MR1/M7 355 410 55 446 539 93 6.9 a M7:Q108K:K40H:T53A:R58L:Q38F:Q4F. As shown above, in contrast to FR1, binding to M7 does not lead to imine to iminium ESPT. Additionally, excitation of the PSB results in a single emission of the PSB, and no hydroxyl group photoacidity was observed. We presume that MR1 can function as a strong photoacid; however, its iminium pKa with the same mutants is generally lower than ThioPhenol, which results in primary SB and residual PSB formation. Clearly, the emission due to photoacid activity of Phenol-SB complexes is blue-shifted in comparison with Phenol-PSB photoacids, simply because the former absorbs at about 350 nm vs. PSB absorption in the range of 470-500 nm. Therefore, future studies could be focused on enhancing the iminium pKa of MR1-hCRBPII complexes to benefit from its potential as a bright and far-red emitting fluorescent tag for deep tissue imaging applications. 185 III.7.2.C. Developing protein-based double ESPT systems with MR1 Thus far in this section, we have discussed the photobasic and photoacidic properties of MR1 complexes. The following describes our attempts to develop the double ESPT system with MR1 complexes. In the first step, we measured the physical characteristics of the MR1/M4 complex. M4 (Q108K:K40E:T53A:Q38F:Q4F:R58H) mutant has yielded the highest ΦDESPT with ThioPhenol, and we sought to investigate whether the designed mutant can activate the ESPT process on both ends of bound MR1 (imine and the hydroxyl group) as well (Table III-24). Table III-25. Comparison of spectroscopic properties of MR1 in complexation with M3 and M4 mutants. high iminium and mutants SS Entry hCRBPII mutant λabs λem Φa (nm) 1 Q108K:K40E:T53A:Q38F:Q4F:R58L (M3) 357 502 145 0.26 2 Q108K:K40E:T53A:Q38F:Q4F:R58H (M4) 357 503 146 014 a Absolute quantum yield was measured on a Quantaurus-QY. As shown above, the addition of R58H mutation does not change the maximum emission wavelength; however, in contrast to the narrow emission spectrum with M3 mutant (entry 1, see Figure III-30), MR1/M4 complex yields a wide emission spanning 370 nm to 780 nm (Figure III-36). Such a broad emission spectrum indicates that MR1/M4 complex mainly acts as a photobase, with minimum photoacid activity of the hydroxyl group upon excitation. 186 Apparently, contrary to ThioPhenol, the addition of R58H alone cannot deprotonate the hydroxyl group in the excited state. Thus, we attempted to mutate amino acids surrounding the MR1 hydroxyl group to proton acceptor residues. SB-OH PSB-OH PSB-O Figure III-36. The absorption and emission spectra of Q108K:K40E:T53A:Q38F:Q4F:R58H/MR1 complex measured at neutral pH. As is illustrated in Figure III-38, F16 is one the closest residues to the hydroxyl group, 3.7 Å, and studies with MR0 showed that its mutation to tyrosine could substantially increase the ΦDESPT (see Table III-4). Besides, Q38Y mutation enhanced the photoacidic properties of MR1 complexes (see Table III-22). Therefore, we sought to explore the effect of F16Y and Q38Y mutations on the ΦDESPT (Table III-25). 187 As shown below, the addition of F16Y and its combination with Q38Y mutation improved the ΦDESPT considerably; the small bump in the emission spectrum of MR1/M4 has grown to a great extent (Figure III-37). For both mutants, the emission spectrum shows two maxima. The blue-shifted emission peak at 490 nm corresponds to the Phenolate-SB complex; the emission of the Phenol-PSB complex is usually more red-shifted, and as in MR1/M3 complex, is slightly beyond 500 nm. Table III-26. Spectroscopic change as the result of F77Y and Q38Y mutations. high iminium and mutants Entry hCRBPII mutant a λabs λem SS (nm) ΦDESPT 1 KEAFF:R58H:F16Y 357 490/603 133/246 0.45 2 KEAFF:R58H:F16Y:Q38Y 355 489/618 134/263 0.54 a KEAFF equals to Q108K:K40E:T53A:Q38F:Q4F. SB-OH PSB-O SB-O Figure III-37. The absorption and emission spectra of Q108K:K40E:T53A:Q38F:Q4F:R58H:F16Y:Q38Y/MR1 complex measured at neutral pH. 188 Furthermore, the red-shifted emission peak is correlated with the double ESPT product, the ThioPhenolate-PSB complex. Noteworthy, the highest ΦDESPT and the largest Stokes shift, 263 nm, was obtained upon the addition of both F16Y and Q38Y mutations to the M4 mutant (entry 2). Q38F A33 F16 T29 M77 Figure III-38. Docked MR1 in the crystal structure of Q108K:K40L:T51V:T53S:R58W-hCRBPII/ThioFluor, and the closest residues around the hydroxyl group. Next, we sought to investigate whether it is feasible to suppress the Phenolate-SB or Phenol-PSB emission peaks through mutation of the surrounding residues. We envisioned developing a complex that emits only as the double ESPT process (Table III-26). Unfortunately, there is no crystal structure available from MR1 complexes to learn about the exact distances between the hydroxyl and surrounding residues. However, studies with ThioPhenol showed a water- 189 mediated hydrogen bonding network between the T29 and the chromophore’s hydroxyl group and that the introduction of T29Y mutation improved the ΦESPT for the resulted photoacid emission (see Figure II-27 and Table II-25). Table III-27. Spectroscopic change as the result of the mutation of residues surrounding the MR1 hydroxyl group. high iminium and mutants Entry hCRBPII mutant a λabs λem SS (nm) ΦDESPT 1 KEAFF:R58H:T29Y 357 501/616 144/259 0.41 2 KEAFF:R58H:F16Y:T29Y 358 493/608 135/250 0.53 3 KEAFF:R58H:M20Y 358 500 142 0.25 4 KEAFF:R58H:F16Y:M20Y 353 489 136 0.18 5 KEAFF:R58H:F16Y:A33M 355 492 137 0.21 6 KEAFF:R58H:F16Y:T51M 357 485/591 128/234 0.46 7 KEAFF:R58H:F16Y:T29Y:Q38Y 356 485 129 0.36 a KEAFF equals to Q108K:K40E:T53A:Q38F:Q4F. The addition of M20Y and A33M in combination with F16Y reduced the ΦDESPT dramatically and mainly resulted in Phenol-PSB or Phenolate-SB complexes emission in the range of 485 nm to 505 nm (entries 3, 4, and 5). As described in Section III.5.1, the introduction of T51M facilitates the deprotonation of the hydroxyl group and increases the ΦDESPT with ThioPhenol. The same effect was observed with MR1 complexes, as the addition of T51M mutation to Q108K:K40E:T53A:Q38F:Q4F:R58H:F16Y template enhanced the double ESPT quantum yield to more than 45% (entry 6). However, the highest ΦDESPT in this table was obtained with the addition of both F16Y and T29Y mutations (entry 2). 190 Nonetheless, none of the newly designed mutants could exceed the ΦDESPT acquired with F16Y:Q38Y. Thus, we chose to investigate if the addition of F16Y, Q38Y, and T29Y mutations altogether would further facilitate the deprotonation of the hydroxyl group and enhance the ΦDESPT accordingly (entry 7). The resultant mutant, KEAFF:R58H:F16Y:T29Y:Q38Y, showed lower ΦDESPT. Presumably, the presence of three close tyrosine residues leads to a steric clash, and tyrosine residues turn away from the cavity and further from the hydroxyl group to relieve the steric hindrance. To summarize our research about developing the double ESPT system with MR1 complexes, we noted that the introduction of proton acceptor residues such as tyrosine at positions F16, Q38, or T29 could enhance the ΦDESPT to more than 50%. However, as described earlier in this section, the double ESPT emission with MR1 complexes is associated with the emission of single ESPT of imine or the hydroxyl group products, Phenol-PSB and Phenolate-SB, respectively. Our attempts to suppress the blue-shifted emission of unwanted excited-state proton transfer were not fruitful up to this point with the handful of protein mutants we tried for this study. As solution studies showed, the free aldehyde and Phenol-SB of MR1 have absorption around 360 nm, close to the Phenol-SB complex with protein, and their emission tails reaching past 600 nm (see Section III.7.2.A). Therefore, controlling the ESPT process selectivity through engineering the hCRBPII mutants would result in a narrow, red-shifted double ESPT emission essential to prevent the 191 emission from free MR1 or unspecific imine or iminium bindings. The above results suggest that MR1 has a great potential for fluorescent imaging as fusion protein tag. III.8 ThioPhenol structure modification and its application in multicolor imaging Our studies indicate that ThioPhenol is the most selective chromophore for the ESPT processes; its complexes can act as a photobase, photoacid, or both upon single excitation, depending on the protein mutant. However, its brightness needs to be improved compared to commercially available dyes such as BODIPY, cyanines, and rhodamines, to name a few. Basically, aside from fluorescence and phosphorescence, non-radiative processes such as internal conversion and intersystem crossing are the primary mechanisms responsible for the excited state relaxation. Most current theories suggest that a restriction in the rotational freedom of a fluorophore will lead to an increase in quantum yield and, consequently, fluorescence brightness. Hence, we sought to investigate whether rigidifying the ThioPhenol structure by preventing the rotation around the carbon-carbon single bond would enhance its brightness. Mr. Mehdi Moemeni successfully designed a synthetic path to cyclize and rigidify the ThioPhenol structure (Figure III-39a). The synthetic procedure of newly designed chromophore Cyclized ThioPhenol (CyThioPhenol) and all other chromophore structures discussed in this thesis are described in Section IV.7. 192 III.8.1 Spectroscopic properties of CyThioPhenol in solution CyThioPhenol is an unknown chromophore structure, and same as previous chromophore structures discussed, it is essential to characterize CyThioPhenol in solution before moving into the protein environment (Figure III- 39b). a. H O S O Preventing the rotation HO HO S via cyclization ThioPhenol CyThioPhenol b. Figure III-39. a. Structures of ThioPhenol and CyThioPhenol. b. Absorbance (left) and emission (right) spectra of CyThioPhenol in different solvents. The absorption wavelength of the cyclized structure shows ~30 nm bathochromic shift as compared to ThioPhenol, changing in a small range of 16 nm (from 408 nm to 424 nm). In addition, the molar extinction coefficient of 193 CyThioPhenol is larger in all solvents (see Table II-1 and Table III-27). On the other hand, the average emission wavelength is blue-shifted by about 13 nm upon cyclization of the ThioPhenol structure, leading to smaller Stokes shifts. Furthermore, the fluorescence quantum efficiency has increased from 1 to 5% in ethanol and DMSO. Noteworthy, this structure shows low solubility in aqueous solutions resulting in aggravation and less intense absorption and emission peak. However, in contrast to ThioPhenol, the emission spectrum in PBS buffer shows two maxima presumably the red-shifted one at 550 nm corresponding to the deprotonated hydroxyl group in the excited state (Table III-27, entry 5). Table III-28. Spectroscopic characterization of CyThioPhenol in various solvents. ε Entry Solvent λabs (nm) λem (nm) SS (nm) Φa (M-1.cm-1) 1 Toluene 416 473 57 42,482 0.01 2 Ethyl acetate 408 466 58 41,594 0.01 3 Dimethyl sulfoxide 421 455 34 39,620 0.05 4 Ethanol 424 462 38 39,619 0.05 5 PBS buffer 423 479/550 56/127 6,935 0.015 a Absolute quantum yield was measured on a Quantaurus-QY. Evidently, CyThioPhenol is not a solvatochromic chromophore; there is no correlation between the emission wavelength and polarity of the solvents, and the emission wavelength spans a narrow range of 24 nm (from 455 nm to 479 nm if the hydroxyl group ESPT is not considered). 194 To mimic the binding in the protein, CyThioPhenol was coupled with n- butyl amine in ethanol. The sample was acidified and basified to characterize the corresponding PSB and deprotonated hydroxyl group in the ground state (Table III-28, and Figure III-40). Table III-29. Spectroscopic characterization of CyThioPhenol and derivatives. ε Entry Compound λabs (nm) λem (nm) SS (nm) Φa (M-1.cm-1) 1 Free Aldehyde 424 462 38 39,619 0.05 2 Phenol-SB 397 460/550 63/153 45,860 0.11 3 Phenol-PSB 499 580 81 62,732 0.04 4 Phenolate-SB 448 555 107 49,538 0.12 a Absolute quantum yield was measured on a Quantaurus-QY. a. b. Figure III-40. Absorbance (left) and emission (right) spectra of CyThioPhenol and derivatives: Phenol-SB, Phenol-PSB, and Phenolate-SB. 195 As shown above, due to the less electron withdrawing nature of the imine as compared to the aldehyde, the absorption wavelength of the Phenol-SB is blue- shifted by 27 nm. Excitation of the Phenol-SB leads to a two maxima emission spectrum at 460 nm and 550 nm. The red-shifted emission peak results from the deprotonation of the hydroxyl group upon excitation and leads to a Stokes shift of more than 150 nm. The maximum emission wavelength of Phenolate-SB obtained upon basifying the solution with 1M NaOH at 555 nm verifies this observation. The most red-shifted absorption and emission wavelength, 499 nm and 580 nm, respectively, results from Phenol-PSB acquired via acidification of the sample with concentrated hydrochloric acid (aq.) solution. As described in Chapter II, we cannot characterize the Phenolate-PSB since it is impossible to produce it in solution. However, we anticipate a substantial enhancement in quantum yield upon forming this complex in the engineered protein pocket due to the formation of a strong ICT system between the alkoxide and the iminium. Furthermore, solution studies show that the emission quantum yield enhances more than twice upon deprotonation of the hydroxyl group (compare phenolate-SB to free aldehyde), which forms a rather weak push-pull system as the result of the less electron-withdrawing effect of SB as compared to the PSB (Figure III-41). Theoretically, it is not possible to deprotonate the hydroxyl group in neutral pH due to the high pKa of the phenol moiety (>9). Additionally, Phenol-SB and Phenol-PSB are barely emissive beyond 650 nm. Therefore, we sought to pursue 196 the development of CyThioPhenol-hCRBPII complexes as a no-wash background-free fluorescent tag. H N N O O S S Phenolate-PSB Phenolate-SB Strong ICT Weak ICT Figure III-41. Schematic comparison of ICT systems in Phenolate-PSB and Phenolate-SB complexes of CyThioPhenol. III.8.2 Developing CyThioPhenol photoacidic complexes There is no crystal structure of any CyThioPhenol-hCRBPII complexes up to this point. However, studies with ThioPhenol demonstrated that mutating a few residues around the hydroxyl group to proton acceptor residues, such as histidine, tyrosine, and cysteine, would enhance the deprotonation of the hydroxyl group and ΦESPT. Hence, we chose to measure the photophysical properties upon adding those mutations (Table III-29). Q108K:K40L:T51V:T53S:R58H template is retained in all mutants tested as screening different amino acids at position 58, including retention of R58, indicated that histidine yields the highest ΦESPT (see Table II-3). ΦESPT is defined as the fraction of total fluorescence from the excited state ThioPhenolate-PSB, which is obtained as explained in Section II-7. 197 In the first step, we sought to investigate the effect of key mutations, F16Y, A33H, A33Y, and L77Y on the ΦESPT. Upon testing with the first three mutants, CyThioPhenol showed higher iminium pKa and ΦESPT as compared with the same protein variants binding ThioPhenol. In addition, it appears that the concentration of the deprotonated hydroxyl group or Phenolate-PSB complex in the ground state is higher with the cyclized structure. Table III-30. Spectroscopic change as the result of the addition of tyrosine and histidine residues around the hydroxyl group. Entry hCRBPII mutant a λabs λem pKa ΦESPT 1 KLVSH:Y19W:A33Y:F16Y 562 598/665 7.3 0.24 2 KLVSH:A33H:L77Y 563 660 6.9 0.73 3 KLVSH:F16Y:L77Y 560/628 650 7.1 > 0.99 a KLVSH is equal to Q108K:K40L:T51V:T53S:R58H. As shown above, the highest ΦESPT was obtained upon the addition of F16Y:L77Y mutations (entry 3). Interestingly, the absorption spectrum of this mutant measured in neutral pH shows three maxima at 382 nm, 560 nm, and 628 nm, which are correlated with Phenol-SB, Phenol-PSB, and Phenolate-PSB, respectively (Figure III-42a). Therefore, we measured the emission spectrum upon excitation at both 560 nm and 628 nm to verify the accuracy of this statement, and as expected, both excitations result in the same emission. Interestingly, even excitation of the SB leads to residual Phenolate-PSB formation showing a substantial Stokes shift of 268 nm (Figure III-42b). Besides, the complex's 198 absorption and emission spectra were monitored upon both acid and base titrations to characterize this system (Figure III-42 c, d). a. b. SB-OH PSB-O PSB-OH c. d. Figure III-42. a. The absorption and b. the emission spectra of KLVSH is equal to Q108K:K40L:T51V:T53S:R58H:F16Y:L77Y/CyThioPhenol complex measured in neutral pH 7.3. c, d. The absorption and emission spectra of the same complex screened upon acidification pH 5.8 and basification pH 8.3 of the solution. 199 As shown in Figure III-42, acidification of the solution leads to a significant increase in Phenol-PSB absorption and emission. Furthermore, clearly due to the high pKa of the hydroxyl group, the majority of Phenolate-PSB is protonated in pH 5.8, resulting in the disappearance of its absorption peak (compare the blue line and the red line). On the contrary, the concentration of Phenolate-PSB increases upon basifying the solution. However, unfortunately, this protein complex was not stable in more basic environments, but we presume that in higher pH values, the concentration of Phenolate-PSB would decrease, and eventually, the Phenolate- SB will be the single species present in solution. Nonetheless, for photoacids, the iminium pKa should be high enough to produce maximum PSB in order to form a strong ICT system upon deprotonation of the hydroxyl group in the excited state. Our previous studies show that L117E/D mutations effectively enhance the iminium pKa through electrostatic interactions between the iminium and the aspartic/glutamic acid side chains. Unfortunately, as described in Chapter II, the addition of these mutations prevents the ESPT of the hydroxyl group. Upon extensive experiments, we realized that L117C gives the best results in terms of increasing the pKa and maintaining the photoacidic properties of the complex. Thus, we sought to explore whether the addition of L117C mutation can increase the pKa with the cyclized structure. Additionally, we measured the photophysical properties of CyThioPhenol upon the addition of T29Y, T51C, T53C mutations, the same residues that yielded the highest ΦESPT with ThioPhenol (Table III-30). 200 To our delight, the addition of L117C mutation increased the iminium pKa without preventing or reducing the ΦESPT. Notably, the average iminium pKa with CyThioPhenol is slightly higher than ThioPhenol leading to minimal SB concertation. Interestingly, the least amount of ΦESPT was obtained upon binding to Q108K:K40L:T51C:T53S:R58H:Y19W:A33H:F16Y:T29Y:L117C and Q108K:K40L:T51V:T53C:R58H:Y19W:A33H:F16Y:T29Y:L117C mutants (entries 3 and 5), which yielded the highest ΦESPT with ThioPhenol. These results prove that L77Y mutation is vital for ESPT of the hydroxyl group, as all other mutants containing this mutation result in higher ΦESPT. Table III-31. Spectroscopic change as the result of the addition of L117C, T29Y, T51C and T53C mutations. Entry hCRBPII mutant λabs λem pKa ΦESPT Φ 1 KLVSH:Y19W:A33H:F16Y:L77Y:L117C a 552/626 653 9.1 > 0.99 0.31 2 KLVSH:A33H:L77Y:L117C 558 658 9.2 0.54 0.33 3 KLCSH:Y19W:A33H:F16Y:T29Y:L117C b 548/631 585 8.9 0.18 0.30 4 KLVCH:Y19W:A33H:L77Y:L117C c 551 666 10 0.84 0.30 5 KLVCH:Y19W:A33H:F16Y:T29Y:L117C 550 582 9.2 0.20 0.36 6 KLVCH:Y19W:A33H:F16Y:L77Y:L117C 549 652 9.3 > 0.99 0.35 7 KLVCH:Y19W:F16Y:T29Y:L77Y:L117C 549 647 9.8 > 0.99 0.32 8 KLVCH:Y19W:A33H:F16Y:T29Y:L77Y:L117C 548 650 9.9 > 0.99 0.35 a KLVSH is equal to Q108K:K40L:T51V:T53S:R58H. b KLCSH is equal to Q108K:K40L:T51C:T53S:R58H. c KLVCH is equal to Q108K:K40L:T51V:T53C:R58H. 201 Moreover, comparing the protein mutants and the ΦESPT in Table III-30 indicates that F16Y is also essential in facilitating the hydroxyl group's deprotonation. entries 2 and 4 contain the L77Y mutation but lack F16Y, which leads to the second-lowest ΦESPT values. Nonetheless, substitution of serine with cysteine in entry 4, Q108K:K40L:T51V:T53C:R58H:Y19W:A33H:L77Y:L117C, leads to higher ΦESPT, 84% vs. 54%, and thus Q108K:K40L:T51V:T53C:R58H was retained for further protein engineering. Figure III-43. The absorption spectra of Q108K:K40L:T51V:T53C:R58H:Y19W:F16Y:T29Y:L77Y:L117C/CyThioPhenol complex recorded upon basifying the solution from pH 7.3 to pH 9.5. All other three protein mutants containing F16Y and L77Y along with T53C resulted in a pretty narrow emission spectrum corresponding to Phenolate-PSB complex and ΦESPT more than 99% (entries 6-8). However, the highest soluble protein expression yield was obtained with Q108K:K40L:T51V:T53C:R58H:Y19W:A33H:F16Y:L77Y:L117C (M6) mutant, 202 entry 6. Additionally, the red-shifted shoulder corresponding to the ThioPhenolate-PSB complex is the smallest with this mutant. Hence, this mutant was chosen for live-cell imaging experiments. Noteworthy, CyThioPhenol complex with Q108K:K40L:T51V:T53C:R58H:Y19W:F16Y:T29Y:L77Y:L117C mutant (entry 7) was more stable in basic pH, and we were able to monitor the absorption spectra change upon base titration. As expected, in higher pH values, all Phenol-PSB is converted to Phenolate-PSB, but with increasing pH values, the Phenolate-PSB concertation decreases upon conversion to the Phenolate-SB complex (Figure III- 43). III.8.2. A. Kinetic measurements of CyThioPhenol binding hCRBPII Photoacid mutants Binding kinetics were measured prior to confocal imaging experiments to explore the proper incubation time; 20 µM protein in PBS buffer at neutral pH 7.3 was incubated with 0.5 equivalent of the ligand at 23 °C, and the increase in absorbance of the corresponding PSB at its 𝜆max was recorded over time. Collected data points were fitted with a second-order rate equation considering multiple reagents protein and the ligand with non-equal concentrations (Figure III-44). The equation and detailed fitting process are described in Section IV.3.5. Compared to ThioPhenol binding photoacid mutants, the half-time of the cyclized structure binding to M6 is longer. However, we envisioned that even longer incubation times would not lead to the fluorescent background as the 203 fluoresce quantum yield of CyThioPhenol significantly increases upon binding the target mutant as compared to the free aldehyde or non-specific bindings (see Table III-30 and Table III-28). The next section of this chapter aims to demonstrate this probe’s utility in live-cell imaging; most importantly, CyThioPhenol cell permeability and its efficient target binding are shown. PSB Formation k = 285 M-1.min-1 t1/2 = 175.5 min R2 = 0.999 Figure III-44. Rate of CyThioPhenol/M6 PSB formation fitted to second-order kinetics with 20 μM protein and 10 μM CyThioPhenol. Plotted is the concentration of free chromophore vs. time. III.8.2. B. Visualization of hCRBPII/CyThioPhenol in mammalian cells Next, we sought to probe the performance of the engineered hCRBPII/CyThioPhenol photoacid complexes as a no-wash live-cell imaging system. As described earlier in this section, the mutant Q108K:K40L:T51V:T53C:R58H:Y19W:A33H:F16Y:L77Y:L117C (M6) was chosen for these studies. As with this mutant, the iminium pKa is high enough (9.3, Table 204 III-30, entry 6) to result in mainly PSB formation, the ΦESPT (>99%), and fluorescence quantum yield (0.35) is the highest with this mutant. In addition, the concentration of ThioPhenolate-PSB in the ground state was negligible, with M6 leading to a single peak for PSB absorption. Furthermore, the protein expression yield with the M6 mutant is relatively high, and it only expresses in the monomeric form. The absorption and emission wavelength of CyThioPhenol/M6 plus its pKa titrations are depicted in Figure III-45. Additionally, all spectroscopic properties of CyThioPhenol/M6 are summarized in Table III-31. pKa= 9.3 R2 = 0.999 Figure III-45. Spectroscopic properties of Q108K:K40L:T51V:T53C:R58H:Y19W:A33H:F16Y:L77Y:L117C mutant with CyThioPhenol including UV-Vis and fluorescence spectra (left) and the pKa titration (right). 205 The spectroscopic properties of CyThioPhenol and ThioPhenol as protein-based photoacidic fluorescent tags are compared in Table III-31. The total brightness has enhanced more than 2.5 times with the cyclized structure, although the binding rate has decreased. Table III-32. Spectroscopic properties of CyThioPhenol/M6 complex compared with ThioPhenol/M3 complex. e k t1/2 b Complex λabs λem ΦESPT Φa pKa (M-1.cm-1) (M-1.min-1) (min) CyThioPhenol/M6 549 652 38,024 > 0.99 0.35 9.3 285 175.5 ThioPhenol/M3 517 679 33,743 > 0.99 0.15 9.8 1467 34 a Absolute quantum yield was measured on a Quantaurus-QY. b Half-life based on the rate constant obtained from second-order rate fitting; measured at 23 °C with 20 μM protein and 0.5 equiv ThioPhenol at pH 7.2. M6: Q108K:K40L:T51V:T53C:R58H:Y19W:A33H:F16Y:L77Y:L117C. M3: Q108K:K40L:T51V:T53C:R58H:Y19W:A33H:F16Y:T29Y:L117C. In the next step, we sought to explore the performance of CyThioPhenol as a no-wash tag for in vivo confocal imaging experiments. Thus, the M6-hCRBPII mutant was cloned into the same vector described in Chapter II, pFlag-CMV2 vector containing EGFP as an internal standard to assess the specificity of the fluorescent signal. This vector was used for whole-cell labeling; additionally, to target M6 in the cytosol, the signaling peptide NES (nuclear export sequence) was fused to the C-terminus of M6, as illustrated in Figure III-46. 206 HeLa cancer cells were then transiently transfected with the fused constructs to express the corresponding protein. Transfected cells were incubated with 5 μM CyThioPhenol overnight at 37 °C. The cells were then directly subjected to confocal imaging without any washing steps prior to confocal imaging experiments. EGFP pFlag-CMV2- EGFP-hCRBPII-NES hCRBPII AmpR Cytosol Signaling Peptide (NES) Figure III-46. Schematic map of EGFP-hCRBPII-NES fusion construct. NES: nuclear export sequence. The green fluorescence observed upon excitation at 488 nm verifies the successful cell transfection and the fusion protein expression (Figure III-47, the green channel). The stained cells were excited with a 559 nm laser, and the far- red emission was collected with a 600 nm to 700 nm bandpass for the red channel. Interestingly, even after overnight incubation of CyThioPhenol with cells (>12 hours), no unspecific red fluorescent signal was observed, confirming the selectivity of this probe (Figure III-47, the red channel). As discussed in Section III.8.1, free CyThioPhenol and off-target imine or iminium formation do not lead to the fluorescent background. First, these species' (Phenol-SB and Phenol-PSB) total fluorescence quantum yields are much lower than the imaging complex. 207 Second, only a minor part of their emission is beyond 600 nm making no-wash imaging feasible. EGFP-NES CyThioPhenol/hCRBPII-NES Ex: 488 nm Ex: 559 nm Em: BP 500 nm- 550 nm Em: BP 600 nm- 700 nm DIC Ex: 488 nm BP 500 nm- 550 nm Figure III-47. Confocal imaging of labeled HeLa cells expressing EGFP-hCRBPII- NES. NES: nuclear export sequence. Cells were stained with 5 µM CyThioPhenol and incubated at 37 °C overnight. Cells were not washed before imaging. 208 III.8.3 Developing the double ESPT systems with CyThioPhenol-hCRBPII complexes In the previous section, we showed the application of CyThioPhenol as a fluorescent photoacid. The successful application of this novel structure in no-wash background-free imaging inspired us to examine imaging with a double ESPT systems. In the first step, we measured the photophysical properties with M3, the photobase mutant that yields the highest ΦESPT for the ESPT of imine to iminium. The obtained results are compared with ThioPhenol binding the same mutant (Table III.32). Table III-33. Spectroscopic properties of ThioPhenol and CyThioPhenol binding M3 mutant. Entry Complex λabs λem SS (nm) ΦESPT Φa 1 ThioPhenol/M3 b 370 532 162 0.98 0.05 2 CyThioPhenol/M3 398 541 143 > 0.99 0.21 a Absolute quantum yield was measured on a Quantaurus-QY. b M3 mutation: Q108K:K40E:T53A:Q38F:Q4F:R58L. As shown above, the bathochromic shift in the absorption wavelength (28 nm) is less than for the emission wavelengths (9 nm), leading to a smaller Stokes shift for the cyclized structure. Nonetheless, the blue-shifted absorption wavelength of ThioPhenol bound to photobase or double ESPT mutants limits its application for live-cell imaging. The most blue-shifted laser confocal microscopes are equipped with a 400 nm laser beam that can excite less than 50% of the corresponding complex and reduces the tag's total brightness (Figure III-48). 209 Interestingly, CyThioPhenol has a 15-fold enhancement in emission, and the fluorescence quantum efficiency is significantly increased (from 0.05 to 0.21). Note the emission intensities of CyThioPhenol/M3 (left) and ThioPhenol/M3 (right). a. b. 370 nm 532 nm 398 nm 541 nm Figure III-48. The absorption and emission spectra of a. ThioPhenol/M3 (red lines) and b. the absorption and emission spectra of CyThioPhenol/M3 complex (blue lines). As a control experiment, the photophysical properties were measured upon binding to Q108K:K40L:T53A:Q38F:Q4F:R58L, wherein the proton source, the glutamic acid at position 40, is substituted with leucine. As expected, the maximum emission wavelength blue-shifted to 471 nm corresponding to the Phenol-SB complex verifying the R58H as the proton donor source. However, the broad 210 emission spectrum indicates that the ESPT process is not entirely stopped with the K40L mutation (Figure III-49). 398 nm 421 nm Figure III-49. The absorption and emission spectra of Q108K:K40L:T53A:Q38F:Q4F:R58L/CyThioPhenol complex measured in neutral pH. Next, we sought to investigate whether adding more acidic residues around the imine bond would facilitate the ESPT process and enhance the emission quantum yield. Notably, residues V62 and I42 are located at a proper distance from imine, 6.7 Å and 5.4 Å, respectively; not too close to protonate the imine in the ground state, and yet not too far to prevent the proton transfer in the excited state (Figure III-50). We also measured the photophysical properties upon mutation to aspartic acid, K40D. Unfortunately, many of the mutants led to insoluble protein expression; expressed proteins’ spectroscopic properties are shown in Table III- 33. 211 Substitution of K40E with K40D mutation in M3 complex, Q108K:K40D:T53A:Q38F:Q4F:R58L, decreases the ΦESPT to less than 50% (entry 1). Additionally, in contrast to M3, this mutant expresses as a mixture of monomer and dimer, which can hamper its further applications in cell imaging. K40E I42 5.4 Å 5.2 Å 6.7 Å V62 Figure III-50. The crystal structure of Q108K:K40E:T53A:R58W:Q38F:Q4F:Y19W/ThioFlour and highlighted V62, I42, and K40E residues. As listed in Table III-33, the addition of V62E and I42E mutations with aspartic acid, glutamic acid, and leucine residues at position 40 (entries 2, 3, and 4, respectively) results in reduced ΦESPT, higher iminium pKa values, and more dimer expressions. Previously Dr. Elizabeth Santos demonstrated that T51V mutation is significantly effective in monomerizing hCRBPII variants.46 Thus, we sought to explore if more monomer formation would have any influence on the 212 ΦESPT. Interestingly, the only mutant, Q108K:K40D:T51V:T53A:R58L:Q38F:Q4F:V62E showing a lower dimer percentage, gave the highest ΦESPT (entry 5). Table III-34. Spectroscopic properties of mutants as the result of the introduction of V62E, I42E, and K40D mutations. Entry hCRBPII mutant λabs λem Dimer% pKa ΦESPT 1 Q108K:K40DALFF a 396 496/558 53 6.4 0.49 2 Q108K:K40DALFF:V62E 397 550 78 5.6 0.73 3 Q108K:K40EALFF:V62E 401 549 69 5.3 0.84 4 Q108K:K40LALFF:I42E 395 446 9 5.2 0.27 5 Q108K:K40D:T51VALFF:V62E 398 539 12 <5 > 0.99 a ALFF is equal to T53A:R58L:Q38F:Q4F These measurements indicate that CyThioPhenol can appear as a photobase, and thus, we sought to develop double ESPT systems with this chromophore. The following aims to describe our attempts to engineer hCRBPII so that CyThioPhenol can act as both photobase and photoacid upon a single photoirradiation. First, we measured CyThioPhenol photophysical properties upon binding M4, Q108K:K40E:T53A:R58H:Q38F:Q4F, the mutant that yielded the highest quantum yield of double ESPT with ThioPhenol (Table III-34). As described earlier in this chapter, M4 was designed simply with the following objectives: K40E 213 is the proton source for imine to iminium ESPT, and R58H acts as the proton acceptor residue for the hydroxyl to alkoxide ESPT process. Table III-35. Spectroscopic properties of ThioPhenol and CyThioPhenol binding M4 mutant. Entry Complex λabs λem SS (nm) ΦESPT Φa 1 ThioPhenol/M4 b 370 612 242 0.98 0.16 2 CyThioPhenol/M4 399 553 154 < 0.50 0.20 a Absolute quantum yield was measured on a Quantaurus-QY. b. M4 mutation: Q108K:K40E:T53A:Q38F:Q4F:R58H. SB-OH PSB-OH PSB-O Figure III-51. Absorption and emission spectra of Q108K:K40E:T53A:Q38F:Q4F:R58H/CyThioPhenol complex measured in neutral pH. Evidently, the maximum emission wavelength of CyThioPhenol/M4 complex at 553 nm is due to the imine ESPT process (see Table III-32). However, its emission spectrum is extremely broad, spanning from 414 nm to 738 nm, 214 indicating that Phenol-PSB is not the only species formed upon excitation (Figure III-51). Therefore, we sought to investigate whether the insertion of proton acceptor residues around the hydroxyl group would enhance the double ESPT emission and consequently give a narrow emission spectrum. Although there is no available crystal structure from any of the CyThioPhenol-hCRBPII complexes, upon studying the ThioPhenol structures, we could identify 7 residues adjacent to the hydroxyl group. We surmised that a nearby basic or proton acceptor residue could abstract the proton from the hydroxyl group, a weak photoacid, and thus, these residues were individually mutated to tyrosine, cysteine, methionine, and histidine (F16, M20, T29, A33, Q38, R58, L77). The photophysical properties of the mutants that led to soluble protein expressions are listed in Table III-35. All mutants show low iminium pKa values (5.0-5.6), leading to SB formation in the ground state. However, upon excitation of the SB at its maximum absorption wavelength (~400 nm), a broad emission spectrum was obtained that is maximized in the range of 550 nm-570 nm, corresponding to the Phenol-PSB complex. Additionally, we observed a small shoulder to the left side of the PSB emission around 485 nm correlating to the Phenol-SB complex for most of the mutants. The absorption and emission spectra of Q108K:K40E:T53A:Q38F:Q4F:R58H:F16Y/CyThioPhenol complex (entry 1) is shown in Figure III-52a. 215 Table III-36. Spectroscopic properties of mutants at positions 16, 20, 29, 33, 38, 58, and 77. Entry hCRBPII mutant a λabs λem SS (nm) 1 KEAFF:R58H:F16Y 399 485/583 86/184 2 KEAFF:R58H:A33H 402 552/618 150/216 3 KEAFF:R58H:A33H:F16Y 400 483/566 83/166 4 KEAFF:R58H:A33M 396 476/553 80/157 5 KEAFF:R58H:A33M:F16Y 400 480/557 80/157 6 KEAFF:R58H:A33C:F16Y 400 483/566 83/166 7 KEAFF:R58H:F16Y:Q38Y 398 486/549 88/151 8 KEAFF:R58H:F16Y:Q38H 401 480/569 79/168 9 KEAFF:R58H:F16Y:Q38Y:T29Y 398 483/553 85/155 10 KEAFF:R58H:M20Y 400 553 53 11 KEAFF:R58H:A33H:L77Y 408 503/555/619 95/147/211 12 KEAFF:R58H:F16Y:Q38Y:L117E 425 475/581 50/156 13 KEAFF:R58Y:F16Y 400 555 155 a KEAFF is equal to Q108K:K40E:T53A:Q38F:Q4F. Unfortunately, none of the designed mutants gave a narrow emission derived from the double ESPT process, which expectedly should appear post 600 nm. However, we noticed a small, red-shifted shoulder next to the Phenol-PSB emission that supposedly corresponds to the Phenolate-PSB complex, the double ESPT process product for most mutants. Particularly for Q108K:K40E:T53A:Q38F:Q4F:R58H:A33H and Q108K:K40E:Q4F:A33H:Q38F:T53A:R58H:L77Y mutants, entries 2 and 11, 216 respectively, the shoulder is more evident and appears around 620 nm. The absorption and emission spectra of the former are shown in Figure III-52b. a. PSB-OH SB-OH b. PSB-OH PSB-O Figure III-52. The absorption and emission spectra of CyThioPhenol upon binding a. Q108K:K40E:T53A:Q38F:Q4F:R58H:A33H and b. Q108K:K40E:Q4F:A33H:Q38F:T53A:R58H:L77Y mutants. 217 Noteworthy, the substitution of histidine with tyrosine in Q108K:K40E:T53A:Q38F:Q4F:R58H:F16Y mutant reduces the red-shifted shoulder correlated with Phenolate-PSB complex and results in a relatively narrow emission spectrum that corresponds to the Phenol-PSB (entry 13). This observation confirms our previous studies that K40H mutation works best to deprotonate the hydroxyl group, but evidently, it is not optimal for this structure. We next sought to explore how changing the residue at position 40 would affect the double ESPT of CyThioPhenol-hCRBPII complexes. Thus, K40 residue was mutated to several different amino acids in the Q108K:K40E:T53A:Q38F:Q4F:R58H template mutant (Table III-36). Table III-37. Spectroscopic properties upon mutating the K40 residue. Entry hCRBPII mutant a λabs b λem SS (nm) pKa 1 K40 392 457/565 65/173 8.2 2 K40E 399 553 154 5.2 3 K40D 395 476/560/623 81/165/228 6.9 4 K40H 396 453/550 57/154 8.4 5 K40R 392 449/562 57/170 5.1 6 K40N 393 473/520 80/127 5.3 a The K40 mutations of M4: Q108K:K40E:T53A:Q38F:Q4F:R58H. b Only the SB maximum absorption wavelength is listed in this table. Same as the template mutant, the designed mutants mainly gave rise to Phenol-SB and Phenol-PSB emission. However, interestingly, excitation of the SB of Q108K:K40D:T53A:Q38F:Q4F:R58H mutant (entry 3) resulted in a triple- 218 maxima emission spectrum. Moreover, this mutant leads to the highest intensity for the Phenolate-PSB and largest stokes shift, 228 nm (Figure III-53). Nonetheless, as shown below, the double ESPT derived emission maximized at 623 nm is associated with other species emission leading to an excessively broad spectrum. Ex 395 nm PSB-O Figure III-53. The absorption and emission spectra of Q108K:K40D:T53A:Q38F:Q4F:R58H/CyThioPhenol complex upon SB excitation. This section showed the successful development of CyThioPhenol as a fluorescent photobase and photoacid upon binding hCRBPII mutants. However, further protein engineering is required to optimize the protein cavity for a double ESPT system. In the light of measuring CyThioPhenol properties upon binding more than 35 different mutants, we realized the position of the proton acceptor 219 residues, such as R58, F16, A33, which were optimized for ThioPhenol, is not well optimized for the cyclized structure. Undoubtedly CyThioPhenol crystal structures will help identify the closet residues that could facilitate its deprotonation. III.8.4 Application of CyThioPhenol in multicolor no-wash live-cell confocal imaging The distinctive absorption and emission features of CyThioPhenol when it is bound to a photoacid vs. photobase mutant are well suited for two-color imaging applications. The photophysical properties of the photobase and photoacid complexes are compared in Table III-37. Table III-38. Spectroscopic properties of CyThioPhenol/M6 complex compared with CyThioPhenol/M3 complex. e k t1/2 b Complex λabs λem ΦESPT Φa pKa (M-1.cm-1) (M-1.min-1) (min) CyThioPhenol/M6 549 652 38,024 > 0.99 0.35 9.3 285 175.5 CyThioPhenol/M3 398 541 36,543 > 0.99 0.21 5.2 N.D 1.6 a Absolute quantum yield was measured on a Quantaurus-QY. b Half-life based on the rate constant obtained from second-order rate fitting; measured at 23 °C with 20 μM protein and 0.5 equiv CyThioPhenol at pH 7.2. M6:Q108K:K40L:T51V:T53C:R58H:Y19W:A33H:F16Y:L77Y:L117C (Photoacid). M3: Q108K:K40E:T53A:Q38F:Q4F:R58L (Photobase). 220 As shown in Figure III-54b, these two complexes' absorption and emission spectra are well separated, 151 nm for the absorption and 111 nm for the maximum emission wavelengths, making it possible to excite and collect the corresponding emission without any fluorescence leaking into another channel. a. pKa Titration pKa= 5.21 R2 = 0.993 b. Figure III-54. a. M3/CyThioPhenol complex pKa titration. b. The absorption (left) and emission (right) spectra of M3 (blue) and M6 (red) mutants upon binding CyThioPhenol measured at pH 7.2. 221 This is primarily due to the pKa values of the photoacid and photobase mutants, M6 and M3, leading to pure PSB and SB formation in the neutral pH (Figure III-52a, see Figure III-45). Noteworthy, the reaction between Q108K:K40E:T53A:Q38F:Q4F:R58L and CyThioPhenol is complete in less than 5 minutes, with a half-life of 1.6 minutes at 23 °C (measured via SB, the only species in neutral pH, absorption spectra monitoring with 20 μM protein with 5 μM CyThioPhenol and plotted to second-order rate kinetics). III.8.4 A. Dual hCRBPII labeling expressed in HeLa cells We sought to examine the performance of dual fluorescent labeling of hCRBPII in live HeLa cells. Thus, M3 and M6 mutants were cloned into the pFlag- CMV2 vector containing EGFP fused to the N-terminus of the proteins. EGFP EGFP pFlag-CMV2- pFlag-CMV2- EGFP-M6-hCRBPII-NES EGFP-M3-hCRBPII-3NLS hCRBPII hCRBPII AmpR AmpR Cytosol Signaling nucleolus Signaling Peptide (NES) Peptide (3NLS) Figure III-55. Schematic map of EGFP-M6-hCRBPII-NES and EGFP-M3- hCRBPII-3NLS fusion constructs. NLS: nuclear localization sequence and NES: nuclear export sequence. To image different cell organelles with two distinct colors, we aimed to express M6 in cytosol and M3 in the cell nucleus by fusing 3NLS and NES 222 localization peptides to the hCRBPII C-terminus, respectively (Figure III-55). The cells were co-transfected with the two vectors and then stained twice with 5 μM of CyThioPhenol each, first for an overnight incubation and second 10 minutes prior to imaging. The cells were then imaged without any washing steps. First, we examine the performance of CyThioPhenol/M3 as a fluorescent photobasic tag (Figure III-56). The cells were transfected solely with the EGFP- M3-hCRBPII-3NLS fusion construct and were imaged only after 5 minutes incubation of 5 µM CyThioPhenol in 37 °C. Cells were excited with a 405 nm laser, and the emission was collected 480 nm-580 nm bandpass (Figure III-56, the red channel). Next, we were able to collect the two colors emission sequentially using the Olympus microscope virtual channel option. First, the CyThioPhenol/M6 complex was excited via a 559 nm laser beam to collect the far-red emission in the 600 nm- 700 nm bandpass, the red channel in Figure III-57. Then we collected the emission of CyThioPhenol/M3 complex in the 480-580 nm window upon its excitation by the 405 nm laser (cyan channel, Figure III-57). Additionally, for both the red and cyan channels, the green fluorescence was collected to explore the CyThioPhenol cell permeabilization and its distribution in each organelle. Fortunately, as shown, no fluorescent background was detected in non-transfected cells. More importantly, none of the complexes showed fluorescence leakage to another organelle. As a proof of concept, this imaging assay successfully demonstrated the feasibility of labeling two sub-cellular compartments with 223 CyThioPhenol and two different hCRBPII tags that act as photoacid and photobase. III.9 Conclusion and future research plans This section described the photoacid and photobasic properties of a novel synthetic dye ThioPhenol and its cyclized derivative, CyThioPhenol, upon binding hCRBPII mutants. Additionally, we designed a protein/ThioPhenol complex for the first time that can act as both photoacid and photobase, able to transfer two protons upon a single photoirradiation called double ESPT. This system provides an unprecedently large Stokes shift (>240 nm), leading to well- separated absorption and emission and successful application in fluorescent labeling of the target protein. Notably, the cyclized derivative shows a higher fluorescence quantum yield and total brightness, and we illustrated its application as a practical fluorescent tag as a photobase. Both chromophores developed as no-wash fluorescent tags with the minimal background because the structure is designed as the ICT, and fluorescence is activated upon binding the target protein. Besides, none of the chromophores display solvatochromic properties, which helps to eliminate washing steps in order to remove unbound chromophores. Nonetheless, our efforts to develop a double ESPT system with CyThioPhenol were not fruitful. Future studies should be focused on developing the double ESPT with brighter cores such as the cyclized derivative or other novel designed structures potential that might find application in medicinal chemistry. 224 Additionally, crystallographic studies and ultrafast spectroscopy should be pursued to understand the mechanism of the double ESPT in the current system and evolve it with other structures. 225 EGFP-3NLS CyThioPhenol/hCRBPII-3NLS Ex: 488 nm Ex: 405 nm Em: BP 500 nm- 550 nm Em: BP 480 nm- 580 nm Green + Red channel DIC + red channel Figure III-56. Confocal imaging of labeled HeLa cells expressing EGFP- hCRBPII-3NLS. NLS = nuclear localization sequence. Cells were stained with 5 mM CyThioPhenol and incubated at 37 °C for 5 min. Cells were not washed before imaging. 226 EGFP CyThioPhenol/hCRBPII-NES Ex: 488 nm Ex: 559 nm Em: BP 500 nm- 550 nm Em: BP 600 nm- 700 nm CyThioPhenol/hCRBPII-3NLS Ex: 405 nm Cyan + Red channel Em: BP 480 nm- 580 nm Figure III-57. Confocal imaging of labeled HeLa cells expressing EGFP- hCRBPII-3NLS and EGFP-hCRBPII-NES. NLS= nuclear localization sequence. NES= nuclear export sequence. Cells were stained with 5 µM CyThioPhenol and incubated at 37 °C for overnight and another 5 µM CyThioPhenol incubated at 37 °C for 5 minutes. Cells were not washed before imaging. 227 REFERENCES 228 REFERENCES 1 Huynh, M. H. V. & Meyer, T. J. Proton-coupled electron transfer. Chem. Rev. 107, 5004-5064, doi:10.1021/cr0500030 (2007). 2 Lee, J., Kim, C. H. & Joo, T. Active Role of Proton in Excited State Intramolecular Proton Transfer Reaction. J. Phys. Chem. A 117, 1400- 1405, doi:10.1021/jp311884b (2013). 3 Joshi, H. C. & Antonov, L. 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Angewandte Chemie-International Edition 57, 16083-16087, doi:10.1002/anie.201810065 (2018). 233 45 Sheng, W. In PhD Thesis. Michigan State University (2019). 46 Santos, E. M. in PhD thesis. Michigan State University (2017). 234 CHAPTER IV: MATERIALS AND METHODS IV.1 Site-directed mutagenesis of hCRBPII and CRABPII Table IV-1. PCR cycling conditions for site-directed mutagenesis. PCR Program Time (min) 1x 94 °C 3:00 94 °C 00:20 30x 3-5 °C below Tm 00:55 72 °C 03:30 1x 72 °C 10:00 1x 4 °C 5:00 Reactant volume DNA (Template Plasmid) 70 ng (x μl) Primer forward 20 pmol (y μl) Primer reverse 20 pmol (z μl) 10 mM dNTP 1 μL DMSO 5 μL 50 mM MgCl2 5 μL 10 x Cloned Pfu Reaction Buffer 5 μL Pfu Turbo DNA Polymerase (2.5 U/μl) 1 μL DI Water 50 – x – y – z – 17 μL All DNA oligonucleotides were purchased from Integrated DNA Technologies (IDT), with melting temperatures (Tm) from approximately 55 °C to 65 °C (depending on primer’s sequence). All E. coli constructs were in the pET- 17b vector (Addgene). The pET-17b plasmid, containing hCRBPII- Q108K:K40L 235 cloned between NdeI and XhoI, was used as a template for mutagenesis of hCRBPII.1,2 The template was used to do single point mutations on CRABPII was the pET-17b plasmid containing wild-type CRABPII cloned between NdeI and EcoRI. Site-directed mutagenesis was conducted via polymerase chain reaction (PCR), using PfuTurbo DNA Polymerase (Agilent) following the specified cycling conditions shown in Table IV-1. The sequences of the forward primers are listed below. It is worth noting that, in all cases, the reverse primer is the reverse complement of the forward primer. All primers correspond to hCRBPII unless otherwise indicated. Q4C: 5’-CG AGG GAC TGC AAT GGA ACC TGG-3’ Q4E: 5’-ACG AGG GAC GAA AAT GGA ACC TGG-3’ Q4F: 5’-G ACG AGG GAC TTC AAT GGA ACC-3’ Q4K: 5’-G ACG AGG GAC AAG AAT GGA ACC TGG G-3’ Q4S: 5’-CG AGG GAC AGC AAT GGA ACC TGG-3’ Q4T: 5’-G ACG AGG GAC ACA AAT GGA ACC-3' Q4Y: 5’-CG AGG GAC TAC AAT GGA ACC TGG GAG-3’ F16C: 5’-GAG AGT AAT GAA AAC TGC GAG GGC TAC ATG-3’ F16C:Y19W: 5’-G AGT AAT GAA AAC TGC GAG GGC TGG ATG-3’ F16E: 5’-GAG AGT AAT GAA AAC GAG GAG GGC TAC ATG-3’ F16E:Y19W: 5’-G AGT AAT GAA AAC GAG GAG GGC TGG ATG-3’ F16F:Y19W: 5’-GAG AGT AAT GAA AAC TTT GAG GGC TGG ATG-3’ 236 F16H: 5’-GAG AGT AAT GAA AAC CAC GAG GGC TAC ATG-3’ F16H:Y19W: 5’-G AGT AAT GAA AAC CAC GAG GGC TGG ATG-3’ F16W: 5’-G AGT AAT GAA AAC TGG GAG GGC TAC ATG-3’ F16W:Y19W: 5’-AGT AAT GAA AAC TGG GAG GGC TGG ATG AAG GCC-3’ F16Y: 5’-G AGT AAT GAA AAC TAT GAG GGC TAC ATG-3’ F16Y:Y19W: 5’-GA GAGT AAT GAA AAC TAT GAG GGC TGG ATG-3’ Y19L: 5’-TTT GAG GGC CTG ATG AAG GCC-3’ Y19W: 5’-C TTT GAG GGC TGG ATG AAG GCC CTG-3’ F16Y:Y19W: 5’-C TAT GAG GGC TGG ATG AAG GCC CTG-3’ Y19W:M20D: 5'-C TTT GAG GGC TGG GAT AAG GCC CTG-3' Y19W:M20E: 5'-C TTT GAG GGC TGG GAG AAG GCC CTG-3' Y19W:M20H: 5’-GAG GGC TGG CAT AAG GCC CTG-3’ Y19W:M20K: 5’-GAG GGC TGG AAA AAG GCC CTG-3’ M20L: 5’-GAG GGC TAC CTG AAG GCC CTG-3’ Y19W:M20R: 5’-GAG GGC TGG AGA AAG GCC CTG-3’ M20Y: 5’-GAG GGC TAC TAC AAG GCC CTG G-3’ T29D: 5'-GAT TTT GCC GAT CGC AAG ATT GC-3' T29E: 5'-GAT TTT GCC GAG CGC AAG ATT GC-3' T29F:A33H: 5'-G GAT ATT GAT TTT GCC TTC CGC AAG ATT CAC-3' T29H:A33W: 5’-GAT TTT GCC CAC CGC AAG ATT TGG-3’ T29K:A33W: 5’-ATT GAT TTT GCC AAG CGC AAG ATT TGG-3’ T29R:A33W: 5’-GAT TTT GCC CGG CGC AAG ATT TGG-3’ T29W:A33H: 5’-GAT TTT GCC TGG CGC AAG ATT CAC-3’ T29Y: 5’-G GAT ATT GAT TTT GCC TAC CGC AAG ATT GC-3’ 237 A33C: 5’-C CGC AAG ATT TGC GTA CGT C-3’ A33D: 5’-ACC CGC AAG ATT GAT GTA CGT CTC-3ʼ A33E: 5ʼ-CGC AAG ATT GAG GTA CGT CTC AC-3ʼ A33H: 5’-ACC CGC AAG ATT CAC GTA CGT CTC-3ʼ A33K: 5’-GCC ACC CGC AAG ATT AAA GTA CGT CTC-3ʼ A33M: 5’-CC ACC CGC AAG ATT ATG GTA CGT CTC AC-3’ A33R: 5’-ACC CGC AAG ATT CGT GTA CGT CTC-3ʼ A33S: 5’-C CGC AAG ATT AGC GTA CGT CTC AC-3’ A33W: 5’-CGC AAG ATT TGG GTA CGT CTC AC-3’ A33Y: 5’-ACC CGC AAG ATT TAC GTA CGT CTC ACT-3’ Q38E:K40L: 5’-GTA CGT CTC ACT GAG ACG CTG GTT ATT GAT CAA-3’ Q38F:K40L: 5’-GTA CGT CTC ACT TTT ACG CTT GTT ATT GAT C-3’ Q38H:K40E: 5’-GCA GTA CGT CTC ACT CAC ACG GAG GTT ATT G-3’ Q38H:K40L: 5’-GTA CGT CTC ACT CAC ACG CTG GTT ATT GAT-3’ Q38P: 5’-GTA CGT CTC ACT CCG ACG AAG GTT ATT G-3’ Q38Q: 5’-GTA CGT CTC ACT CAG ACG AAG GTT ATT GAT-3’ Q38Y:K40E: 5’-GCA GTA CGT CTC ACT TAC ACG GAG GTT ATT G-3’ Q38Y:K40L: 5’-GCA GTA CGT CTC ACT TAC ACG CTG GTT ATT GAT-3’ K40A: 5’-CTC ACT CAG ACG GCT GTT ATT GAT CAA-3’ K40C: 5’-CTC ACT CAG ACG TGC GTT ATT GAT CAA G-3’ Q38F:K40C: 5’-GTA CGT CTC ACT TTT ACG TGT GTT ATT GAT-3’ K40D: 5’-CTC ACT CAG ACG GAT GTT ATT GAT CAA GAT GG-3’ Q38F:K40D: 5’-GTA CGT CTC ACT TTT ACG GAC GTT ATT GAT CAA-3’ K40E: 5’-CTC ACT CAG ACG GAG GTT ATT GAT CAA-3’ 238 Q38F:K40E: 5’-GTA CGT CTC ACT TTT ACG GAA GTT ATT GAT CAA-3’ K40H: 5’-CTC ACT CAG ACG CAC GTT ATT GAT CAA-3’ Q38F:K40H: 5’-GTA CGT CTC ACT TTT ACG CAT GTT ATT GAT-3’ K40K: 5’-CGT CTC ACT CAG ACG AAG GTT ATT GAT CAA GAT-3’ Q38F:K40K: 5’-GTA CGT CTC ACT TTT ACG AAG GTT ATT-3’ Q38F:K40L: 5’-CGT CTC ACT TTC ACG CTG GTT ATT GAT C-3’ Q38F:K40M: 5’-GTA CGT CTC ACT TTC ACG ATG GTT ATT-3’ K40N: 5’-CTC ACT CAG ACG AAC GTT ATT GAT CAA G-3’ Q38F:K40N: 5’-CTC ACT TTT ACG AAC GTT ATT GAT CAA GAT GG-3’ Q38F:K40P: 5’-C ACT TTT ACG CCG GTT ATT GAT CAA GAT GG-3’ K40Q: 5’-CTC ACT CAG ACG CAG GTT ATT GAT CAA G-3’ Q38F:K40Q: 5’-GTA CGT CTC ACT TTT ACG CAA GTT ATT GAT-3’ Q38F:K40R: 5’-GTA CGT CTC ACT TTT ACG CGA GTT ATT GAT-3’ K40S: 5’-CTC ACT CAG ACG TCG GTT ATT GAT CAA GAT GG-3’ Q38F:K40S: 5’-GTA CGT CTC ACT TTT ACG TCT GTT ATT GAT-3’ K40T: 5’-CTC ACT CAG ACG ACG GTT ATT GAT CAA G-3’ Q38F:K40T: 5’-GTA CGT CTC ACT TTT ACG ACA GTT ATT-3’ K40Y: 5’-CTC ACT CAG ACG TAC GTT ATT GAT CAA GAT GG-3’ Q38F:K40Y: 5’-GTA CGT CTC ACT TTT ACG TAC GTT ATT-3’ K40L:I42E: 5’-ACG CTG GTT GAA GAT CAA GAT GGT-3’ K40H:I42F: 5’-ACG CAT GTT TTC GAT CAA GAT GGT GAT-3’ K40L:I42K: 5’-CAG ACG CTG GTT AAG GAT CAA GAT GGT G-3’ K40H:I42P: 5’-ACG CAT GTT CCC GAT CAA GAT GGT-3’ K40H:I42W: 5’-ACG CAT GTT TGG GAT CAA GAT GGT GAT-3’ 239 Q44K: 5’-CT CAG ACG CTG GTT ATT GAT AAG GAT GGT GAT AAC-3’ F49K: 5’-GAT CAA GAT GGT GAT AAC AAG AAG GTA AAA AGC AC-3’ T51C:T53S: 5’-GGT GAT AAC TTC AAG TGC AAA AGC ACT AGC AC-3’ T51E:T53E: 5’-GGT GAT AAC TTC AAG GAG AAA GAG ACT AGC-3’ T51M:T53A: 5’-GGT GAT AAC TTC AAG ATG AAA GCG ACT AGC-3’ T51M:T53S: 5’-GGT GAT AAC TTC AAG ATG AAA AGC ACT AGC AC-3’ T51T:T53S: 5’-GGT GAT AAC TTC AAG ACA AAA AGC ACT AGC AC-3’ T51T:T53A: 5’-C TTC AAG ACA AAA GCG ACT AGC ACA TTC CG-3’ T51V:T53C: 5’-C TTC AAG GTA AAA TGC ACT AGC ACA TTC CAC-3’ T53D: 5ʼ-C TTC AAG ACA AAA GAT ACT AGC ACA TTC CG-3ʼ T53E: 5ʼ-TTC AAG ACA AAA GAG ACT AGC ACA TTC-3ʼ T51V:T53E: 5’-AAC TTC AAG GTA AAA GAG ACT AGC ACA TTC CAC AAC-3’ T51V:T53H: 5’-AAC TTC AAG GTA AAA CAC ACT AGC ACA TTC CAC AAC-3’ T51V:T53S: 5’-AAC TTC AAG GTA AAA AGC ACT AGC ACA TTC CAC AAC-3’ T51V:T53Y: 5’-AAC TTC AAG GTA AAA TAC ACT AGC ACA TTC CAC AAC TAT-3’ T51V:T53S:S55E: 5’-AAG GTA AAA AGC ACT GAG ACA TTC CAC AAC TAT GAT-3’ T51V:T53S:S55H: 5’-AAG GTA AAA AGC ACT CAC ACA TTC CAC AAC TAT GAT-3’ T51V:T53S:S55S :5’-GTA AAA AGC ACT AGC ACA TTC CAC AAC TAT GAT GTG-3’ T51V:T53S:S55Y: 5’-AAG GTA AAA AGC ACT TAC ACA TTC CAC AAC TAT GAT GTG- 3’ R58A: 5'-CT AGC ACA TTC GCG AAC TAT GAT GTG-3' R58C: 5’-GC ACT AGC ACA TTC TGC AAC TAT GAT GTG G-3’ R58E: 5’-CT AGC ACA TTC GAG AAC TAT GAT GTG-3’ R58H: 5’-CT AGC ACA TTC CAC AAC TAT GAT GTG-3’ 240 R58K: 5’-AGC ACA TTC AAG AAC TAT GAT GTG-3’ R58L: 5’-CT AGC ACA TTC CTG AAC TAT GAT GTG-3’ R58M: 5’-CC ACT AGC ACA TTC ATG AAC TAT GAT GTG G-3’ R58Q: 5’-CT AGC ACA TTC CAG AAC TAT GAT GTG-3’ R58R: 5’-C ACT AGC ACA TTC CGC AAC TAT GAT GTG GAT TTC A-3’ R58S: 5’-ACC ACT AGC ACA TTC TCA AAC TAT GAT GTG GAT-3’ R58T: 5’-CC ACT AGC ACA TTC ACG AAC TAT GAT GTG G-3’ R58W: 5’-CT AGC ACA TTC TGG AAC TAT GAT GTG-3’ R58Y: 5’-CT AGC ACA TTC TAC AAC TAT GAT GTG-3’ Y60L: 5’-TTC CGC AAC CTG GAT GTG GAT-3’ V62Y: 5’-C CGC AAC TAT GAT TAC GAT TTC ACT GTTG G-3’ V62F: 5’-C CGC AAC TAT GAT TTC GAT TTC ACT GTT GG-3’ V62Y: 5’-C CGC AAC TAT GAT TAC GAT TTC ACT GTT GG-3’ F64E: 5’-AAC TAT GAT GTG GAT GAA ACT GTT GGA GTA GAG-3’ S76D: 5’-TAC ACA AAG GAC CTG GAT AAC CGG-3ʼ S76E: 5’-TAC ACA AAG GAG CTG GAT AAC CGG-3ʼ S76G: 5’-GAG TAC ACA AAG GGC CTG GAT AAC CGG-3’ S76H: 5’-TAC ACA AAG CAC CTG GAT AAC-3’ S76K: 5’-GAG TAC ACA AAG AAG CTG GAT AAC-3’ S76R: 5’-TAC ACA AAG CGA CTG GAT AAC-3’ S76V: 5’-GAG TAC ACA AAG GTC CTG GAT AAC-3’ L77A: 5ʼ-C ACA AAG AGC GCA GAT AAC CGG C-3ʼ L77C: 5ʼ-G TAC ACA AAG AGC TGC GAT AAC CGG CAT G-3ʼ L77D: 5ʼ-G TAC ACA AAG AGC GAT GAT AAC CGG-3ʼ 241 L77E: 5ʼ-G TAC ACA AAG AGC GAG GAT AAC CGG-3ʼ L77F: 5ʼ-G TAC ACA AAG AGC TTC GAT AAC CGG CAT G-3ʼ L77H: 5’-ACA AAG AGC CAC GAT AAC CGG CAT-3’ S76G:L77I: 5’-C ACA AAG GGC ATC GAT AAC CGG CAT G-3’ L77K: 5’-ACA AAG AGC AAA GAT AAC CGA-3’ L77M: 5’-G TAC ACA AAG AGC ATG GAT AAC CGG C-3’ L77R: 5’-ACA AAG AGC CGG GAT AAC CGG-3’ L77S: 5ʼ-C ACA AAG AGC TCA GAT AAC CGG C-3ʼ L77W: 5’-G TAC ACA AAG AGC TGG GAT AAC CGG CAT G-3’ L77Y: 5’-G TAC ACA AAG AGC TAC GAT AAC CGG CAT G-3’ L93E: 5’-TGG GAA GGT GAT GTC GAA GTG TGT GTG CAA AAG-3’ L93K: 5’-GG GAA GGT GAT GTC AAG GTG TGT GTG-3’ Q108A: 5’-GGC TGG AAG GCC TGG ATT GAG G-3’ Q108L: 5’-GGC TGG AAG CTG TGG ATT GAG G-3’ Q108Q: 5’-C GGC TGG AAG CAA TGG ATT GAG G-3’ Q108A:I110K: 5’-GGC TGG AAG GCC TGG AAG GAG G-3’ Q108Q:I110K: 5’-GGC TGG AAG CAG TGG AAA GAG GGG GAC-3’ I110K: 5’-G AAG CAA TGG AAG GAG GGG GAC AAG-3’ L115E: 5’-GGG GAC AAG GAG TAC CTG GAG C-3’ L117C: 5’-GAC AAG CTG TAC TGT GAG CTG ACC TGT-3’ L117D: 5’-C AAG CTG TAC GAC GAG CTG ACC-3’ L117E: 5’-C AAG CTG TAC GAG GAG CTG ACC-3’ L117H: 5’-GAC AAG CTG TAC CAC GAG CTG-3’ L117M: 5’-GAC AAG CTG TAC ATG GAG CTG ACC TG-3’ 242 L117Q: 5’-C AAG CTG TAC CAG GAG CTG ACC-3’ L117S: 5’-GAC AAG CTG TAC AGC GAG CTG ACC TG-3’ L117T: 5’-C AAG CTG TAC ACA GAG CTG ACC-3' L117Y: 5’-GAC AAG CTG TAC TAC GAG CTG ACC TGT G-3’ L119E: 5ʼ-G CTG TAC CTG GAG GAG ACC TGT GGT GAC-3’ L119D: 5ʼ-G CTG TAC CTG GAG GAT ACC TGT GGT GAC-3ʼ Q128E: 5’-CAG GTG TGC CGT GAG GTG TTC AAA AAG-3’ Q128F: 5’-GTG TGC CGT TTT GTG TTC AAA-3’ Q128H: 5’-CAG GTG TGC CGT CAC GTG TTC AAA AAG-3’ Q128M: 5’-GTG TGC CGT ATG GTG TTC AAA-3’ Q128S: 5’-CAG GTG TGC CGT AGC GTG TTC AAA AAG-3’ Q128T: 5’-CAG GTG TGC CGT ACC GTG TTC AAA AAG-3’ Q128Y: 5’-CAG GTG TGC CGT TAC GTG TTC AAA AAG AAG-3’ F130C: 5’-CAG GTG TGC CGT CAA GTG GAG AAA AAG AAG-3’ F130E: 5’-GC CGT CAA GTG TGC AAA AAG AAG TG-3’ F130H: 5’-GC CGT CAA GTG CAC AAA AAG AAG TG-3’ F130Q: 5’-G TGC CGT CAA GTG CAG AAA AAG AAG TG-3’ F130S: 5’-GC CGT CAA GTG TCC AAA AAG AAG TG-3’ F130T: 5’-GC CGT CAA GTG ACC AAA AAG AAG TG-3’ F130Y: 5’-CAG GTG TGC CGT CAA GTG TAC AAA AAG AAG-3’ V41E (CRABPII): 5’-CC AAG CCA GCA GAG GAG ATC AAA CAG G-3’ V41I (CRABPII): 5’-GCG TCC AAG CCA GCA ATC GAG ATC AAA C-3’ V41Q (CRABPII): 5’-CC AAG CCA GCA CAG GAG ATC AAA CAG-3’ T54D (CRABPII): 5’-C TAC ATC AAA GAC TCC ACC ACC GTG C-3’ 243 R59E (CRABPII): 5’-C ACC ACC GTG GAG ACC ACA GAG-3’ L121N (CRABPII): 5’-GG GAA CTG ATC AAC ACC ATG ACG GCG-3’ L121Q (CRABPII): 5’-GAA CTG ATC CAG ACC ATG ACG GCG-3’ L121W (CRABPII): 5’-GGG GAA CTG ATC TGG ACC ATG ACG-3’ To digest the original template DNA, the crude PCR products were subjected to 20 units of DpnI enzyme (New England Biolabs), and were incubated at 37 °C for one h. The resulting solution (15 μL) was then added to E. coli XL-1 Blue competent cells (Novagen, 50 μL), and incubated on ice for at least 30 min. Subsequently, the solution was spread gently on a Luria broth (LB) agar plate supplemented with 100 μg/mL ampicillin and 12.5 μg/mL tetracycline. The plate was incubated at 37 °C for approximately 20 h. A single and well-isolated colony was then picked from the agar plate and inoculated into 10 mL LB media containing 100 μg/mL ampicillin and 12.5 μg/mL tetracycline. LB media was prepared by adding 10 g tryptone, 10 g yeast extract, and 5 g NaCl to 1 L DI water. The media was autoclaved and cooled to room temperature before use. The inoculated LB media was shaken at 37 °C for approximately 20 h. Cells were harvested via centrifugations at 5000 rpm for 10 min. PCR-amplified DNA was purified by a Promega Wizard Plus SV miniprep DNA purification system (A1330) following the manufacturers’ directions with the exception of using 45 μL of Nuclease-Free water for DNA elution instead of the recommended 100 μL to yield more concentrated DNA product. 244 The isolated plasmids’ concentration was measured via Thermo Scientific NanoDrop™ 1000 Spectrophotometer; the average concentration was 300 ng/μL in the 45 μL solution. A sample containing at least 700 ng (about 8 μL) of purified plasmid was transferred into another Eppendorf tube and sequenced by The Research Technology Support Facility at Michigan State University using a primer corresponding to the T7 promoter for all pET-17b plasmid. IV.2 hCRBPII and CRABPII expression and purification in pET-17b expression plasmids The target gene (100 ng of DNA for 100 μL cell solution) was added to thawed BL21(DE3) pLysS competent cells (Invitrogen™) E. coli competent cells on ice and incubated for 30 min, and subsequently, the cells were gently spread on a Luria broth (LB) agar plate supplemented with 100 μg/mL ampicillin and 27 μg/mL chloramphenicol. The plate was incubated at 37 °C for approximately 12 h. In order to grow a cell culture, a single colony was then inoculated into 1 L terrific broth (TB) media supplemented with 100 μg/mL ampicillin and 27 μg/mL chloramphenicol. TB media was prepared by mixing two solutions and autoclaving them separately. The first solution consists of 12 g tryptone, 24 g yeast extract, and 4 mL glycerol to 900 mL DI water. The second solution was prepared by mixing 2.31 KH2PO4 and 12.54 g of K2HPO4 in 90 mL DI water. Before inoculation, the solutions were mixed. The inoculated culture was shaken at 37 °C at 250 rpm until optical density (OD) at 600 nm was in the range of 0.75 - 0.95; this typically takes 7 to 9 h. Overexpression was induced by adding 1 mL of 1 M isopropyl-β-D- 245 thiogalactopyranoside (IPTG, Gold Biotechnology), resulting in final concentration of 1 mM. The culture was then shaken at 23 °C for 20 h at 225 rpm. The cells were then harvested by centrifugation (5000 rpm, 12 min, 4 °C). The supernatant was discarded, and the cells were resuspended in 50 mL Tris- binding buffer (10 mM Tris, pH=7.8-8.0). The cells were then lysed by sonication (Model 300 V/T Ultrasonic Homogenizer, Biologics Inc, power 60%, 3 min), and treated with DNAase (300 units/ 50 mL suspension) and MgCl2 (0.12 mmol/ 50 mL suspension). The solution was again centrifuged to separate the pellet and supernatant (5000 rpm, 40 min, 4°C). All further protein purification was also conducted at 4 °C. The protein in the supernatant was purified as follow: The supernatant was loaded onto an anion exchange column (Q Sepharose TM Fast Flow resin, GE Healthcare, column diameter: ~ 4 cm; height: ~10 cm), pre-equilibrated with the Tris buffer (10 mM Tris, pH= 7.8-8.0). After binding the protein to the Fast Q anion exchange resin, the column was washed twice with the Tris buffer (100 mL). The protein was eluted with Tris-elution buffer (100 mL, 10 mM Tris, 200 mM sodium chloride, pH=8.0). The eluent from the Fast Q anion exchange column was desalted with Tris buffer using an ultrafiltration cell under nitrogen pressure (~20 psi) equipped with a 10 kDa molecular weight cutoff membrane (Millipore, Regenerated Cellulose membrane, diameter 63.5 mm, NMWL: 10,000, filter code: YM10). The protein was first concentrated to ~ 35 mL and then diluted to 150 mL with Tris buffer. This solution was concentrated again to ~ 30 mL. 246 Further purification was continued with Fast Protein Liquid Chromatography (NGC chromatography system, Biorad), equipped with a column loaded with SOURCE 15Q (Q Sepharose Fast Flow, GE Healthcare) anion exchange resin. The method for FPLC SOURCE Q is shown in Table IV-2. The pH at all steps was set to 8.1% B corresponds to the percent salt, where 100% is equivalent to 1 mM NaCl. Table IV-2. FPLC Source 15Q method. Step Description %B Volume Flow Rate 1. Isocratic flow 0 12 mL 3 mL/min Sample 2. Load sample n/a 2 mL/min volume 3. Isocratic flow 0 10 mL 3 mL/min 4. Gradient flow 0 to 4 12 mL 3 mL/min 5. Isocratic flow 4 20 mL 3 mL/min 6. Gradient flow 4 to 8 15 mL 3 mL/min 7. Isocratic flow 8 20 mL 3 mL/min 8. Gradient flow 8 to 15 15 mL 3 mL/min 9. Isocratic flow 15 40 mL 3 mL/min 10. Gradient flow 15 to 75 10 mL 3 mL/min 11. Isocratic flow 100 20 mL 3 mL/min 12. Isocratic flow 0 35 mL 3 mL/min Table IV-3. FPLC SEC method. Step Description %B Volume Flow Rate 1. Load sample 0 4 mL 1 mL/min 2. Isocratic flow 20 139 mL 1 mL/min 247 Next, all tubes with 40 mM, 80 mM, and 150 mM of NaCl, which have higher intensity for 280 nm peak (green) in comparison to 260 nm peak (purple), were collected. Then the solution was concentrated to 1 mL using a 10 kDa Centriprep centrifugal filter (Millipore, Regenerated Cellulose membrane, NMWL: 10,000). The concentrated sample was then loaded to the Fast Protein Liquid Chromatography (NGC chromatography system, Biorad), equipped with a column loaded with size exclusion chromatography (SEC) Superdex 75 Prep Grade resin (GE Healthcare). The method for FPLC size exclusion is shown in Table IV-3. At all steps, the pH was set to 8.1. IV.3 Protein characterization IV.3.1 UV/Vis and fluorescence spectroscopy Spectroscopic characterizations of purified proteins were carried out using a Cary 300 Bio UV-Visible spectrophotometer (Varian) using 1-cm, 1.0-mL quartz micro cuvettes (Starna Cells). Fluorescence spectroscopy was performed on a Fluorolog®-3 spectrofluorometer (Jobin Yvon, Horiba Scientific) with 1-cm, 3.5-mL quartz cuvettes, or 1-cm, 1.0-mL quartz micro cuvettes (Starna Cells). An entrance slit of 2 nm and an exit slit of 2 nm was used for all measurements. For all experiments, Protein samples (20 μM) were incubated with ligand (0.5 equiv) in 2X PBS buffer and incubated at room temperature until Schiff base (SB) or protonated Schiff base (PSB) formation was complete. This was verified by the protein/chromophore complex UV-Vis spectrum. 248 A stock solution of PBS buffer (10 x) was prepared by dissolving 2.0 g KCl (26 mM), 2.4 g KH2PO4 (17.6 mM), 80 g NaCl (1368 mM), 11.45 g Na2HPO4 (80.7 mM) in 1000 mL DI water. The solution was autoclaved and then diluted to 2 x with autoclaved DI water and filtered before using. IV.3.2 Extinction coefficient determination The extinction coefficients of the proteins were measured at 280 nm, as previously described by Gill and Von Hippel.3 The theoretical extinction (ε Theor ) coefficient is calculated based on the following formula: ε Theor = a × ε Trp + b × ε Tyr + c × ε Cys where a, b and c are the numbers of tryptophans, tyrosines, and cysteine residues, respectively. The extinction coefficients of the three residues were determined at 280 nm previously (ε Trp = 5,690 M-1·cm-1, ε Tyr = 1,280 M-1·cm-1, ε Cys = 120 M-1·cm- 1). The protein absorption was measured at 280 nm under native (2 x PBS buffer) and denaturing (final concentration 6 M guanidine HCl) conditions. The ratio of absorbance intensities under native (Anative) and denaturing (Adenaturing) conditions, multiplied by ε theor yielded ε exp as shown in the following equation. A native ε exp = × ε theor A denaturing 249 The concentration of the protein can be measured via the Lambert-Beer’s equation. where b is the cuvette path length, and c is the concentration of the protein. A native = ε exp × b × c The extinction coefficients of all purified hCRBPII and CRABPII monomers described in this thesis are listed in Table IV-4. All proteins are hCRBPII mutants unless otherwise noted. For the proteins forming a stable dimer, the extinction coefficient of the dimer is calculated as well. (The extinction coefficient values are upon binding ThioPhenol). Table IV-4. Extinction coefficients of hCRBPII and CRABPII mutants. Protein εexp (280 nm) Monomer Dimer Q108K:K40L:T51V:T53S 30,888 Q108K:K40L:T51V:T53S:R58W 33,984 Q108K:K40L:T51V:T53S:R58H 28,504 Q108K:K40L:T51V:T53S:R58E 28,960 Q108K:K40L:T51V:T53S:R58L 29,079 Q108K:K40L:T51V:T53S:R58Q 29,013 Q108K:K40L:T51V:T53S:R58K 28,640 Q108K:K40L:T51V:T53S:R58C 26,929 Q108K:K40L:T51V:T53S:R58S 28,562 Q108K:K40L:T51V:T53S:R58Y 28,466 250 Table IV-4 (cont’d) Q108K:K40L:Y19W:T51V:T53S:R58W 36,880 Q108K:K40L:Y19W:T51V:T53S:R58H 34,571 Q108K:K40L:Y19W:A33W:T51V:T53S:R58W 43,042 Q108K:K40L:Y19W:A33W:T51V:T53S:R58H 37,958 Q108K:K40L:Y19W:A33W:T51V:T53S:R58W:L117E 42,130 Q108K:K40L:Y19W:A33W:T51V:T53S:R58H:L117E 38,408 Q108K:K40L:Q4F:Y19W:T51V:T53S:R58W: L117D 38,135 Q108K:K40L:Q4F:Y19W:T51V:T53S:R58H:L117D 31920 Q108K:K40L:Y19W:T51V:T53S:R58W:L117E 35721 Q108K:K40L:Y19W:T51V:T53S:R58H:L117E 32192 Q108K:K40E:Q4F:Q38F:T53A:R58H 24,391 Q108K:K40E:Q4F:Q38F:T53A:R58L 27,681 Q108K:K40L:Y19W:A33W:T51V:T53S:R58W:L77K:L117E 39, 330 Q108K:K40L:Y19W:A33W:T51V:T53S:R58W:S76R:L117E 39,763 Q108K:K40L:Y19W:A33W:T51V:T53S:R58W:S76H:L117E 38, 892 Q108K:K40L:Y19W:A33W:T51V:T53S:R58W:S76K:L117E 39,995 Q108K:K40L:Y19W:T29H:A33W:T51V:T53S:R58W:L117E 40,434 Q108K:K40L:Q4F:Y19W:T29K:T51V:T53S:R58W:L117D 35,743 Q108K:K40L:Q4F:Y19W:T51V:T53S:R58W:L77H:L117D 36,213 Q108K:K40L:Q4F:Y19W:M20K:T51V:T53S:R58W:L117D 35,893 Q108K:K40L:Q4F:Y19W:M20H:T51V:T53S:R58W:L117D 36,187 Q108K:K40L:Q4F:Y19W:T51V:T53S:R58W:S76H:L117D 37,121 251 Table IV-4 (cont’d) Q108K:K40L:Q4F:Y19W:A33H:T51V:T53S:R58W:L117D 34,862 Q108K:K40L:Q4F:Y19W:A33E:T51V:T53S:R58W:L117D 35,628 Q108K:K40L:Q4F:Y19W:T51V:T53S:R58W:S76E:L117D 36,986 Q108K:K40L:Q4F:Y19W:T29D:T51V:T53S:R58W:L117D 37,334 Q108K:K40L:Q4F:Y19W:T29E:T51V:T53S:R58W:L117D 34,761 Q108K:K40L:A33H:T51V:T53S:R58H 25,741 Q108K:K40L:A33H:T51V:T53S:R58W 30,869 Q108K:K40L:Y19W:A33H:T51V:T53S:R58W:L117E 39,391 Q108K:K40L:Y19W:A33H:T51V:T53S:R58H 29,874 Q108K:K40L:A33E:T51V:T53S:R58H 27,154 Q108K:K40L:A33S:T51V:T53S:R58H 27,480 Q108K:K40L:Y19W:A33S:T51V:T53S:R58H 34,205 Q108K:K40L:Y19W:A33E:T51V:T53S:R58H 34,827 Q108K:K40L:A33Y:T51V:T53S:R58H 29,287 Q108K:K40L:Y19W:A33Y:T51V:T53S:R58H 33,034 Q108K:K40L:Y19W:A33H:T51V:T53Y:R58H 33,718 Q108K:K40L:Y19W:A33W:T51V:T53H:R58H 38,143 Q108K:K40L:Y19W:A33W:T51V:T53Y:R58H 38,497 Q108K:K40L:F16Y:A33H:T51V:T53S:R58H 29,244 Q108K:K40L:F16Y:Y19W:A33Y:T51V:T53S:R58H 35,708 Q108K:K40L:F16Y:T51V:T53S:R58H 30,701 Q108K:K40L:F16Y:Y19W:A33W:T51V:T53S:R58H 34,602 252 Table IV-4 (cont’d) Q108K:K40L:Y19W:A33H:T51V:T53H:R58H 29,754 Q108K:K40L:Y19W:A33W:T51V:T53E:R58H 39,205 Q108K:K40L:T51V:T53S:R58H:L117E 26,669 Q108K:K40L:A33H:T51V:T53S:S55E:R58H 32,006 53,816 Q108K:K40L:F16Y:Y19W:A33H:T51V:T53S:R58H 34,158 Q108K:K40L:F16Y:Y19W:A33H:T51V:T53Y:R58H 32,919 Q108K:K40L:Q38E:T51V:T53S:R58H 25,760 Q108K:K40L:F16E:A33H:T51V:T53S:R58H 29,098 Q108K:K40L:F16E:T51V:T53S:R58H 26,727 Q108K:K40L:F16H:Y19W:A33W:T51V:T53S:R58H 35,303 Q108K:K40L:F16H:Y19W:A33Y:T51V:T53S:R58H 38,777 Q108K:K40L:F16E:Y19W:A33W:T51V:T53S:R58H 39,943 Q108K:K40L:A33H:Q38Y:T51V:T53S:R58H 27,958 Q108K:K40L:F16Y:A33H:I42E:T51V:T53S:R58H 54,179 Q108K:K40L:F16Y:Y19W:A33W:T51E:T53S:R58H 39,061 Q108K:K40L:F16E:Y19W:A33Y:T51V:T53S:R58H 34,119 Q108Q:K40L:T51V:T53S:I110K 30,045 Q108K:K40L:Q38H:T51V:T53S:R58H 25,095 Q108K:K40L:T51V:T53E:R58H 26,199 Q108K:K40L:T51V:T53Y:R58H 28,178 Q108K:K40L:T51V:T53S:S55Y:R58H 54,120 Q108K:K40L:T51V:T53S:S55E:R58H 32,005 53,376 253 Table IV-4 (cont’d) Q108K:K40L:Y19W:A33H:T51V:T53E:R58H 33,433 Q108K:K40L:T51V:T53S:S55H R58H 50,732 Q108K:K40L:F16W:A33H:T51V:T53S:R58H 32,566 Q108K:K40L:Y19W:A33H:Q38E:T51V:T53E:R58H 37,341 Q108K:K40L:F16Y:Y19W:A33H:T51V:T53E:R58H 31,938 Q108K:K40L:F16Y:A33H:T51V:T53E:R58H 27,444 Q108K:K40L:Y19W:A33Y:T51V:T53E:R58H 33,338 Q108K:K40L:A33H:T51V:T53E:R58H 28,006 Q108K:K40L:F16Y:Y19W:A33H:Q38E:T51V:T53S:R58H 31,488 Q108K:K40L:Q38E:T51V:T53E:R58H 27,454 Q108K:K40L:F16Y:Y19W:A33W:T51V:T53E:R58H 38,611 Q108K:K40L:F16Y:A33H:Q38E:T51V:T53S:R58H 29,734 Q108K:K40L:F16Y:Q38E:T51V:T53S:R58H 28,978 Q108K:K40L:F16Y:A33H:T51V:T53S:R58H:L117T 27,933 Q108K:K40L:Q38E::T51V:T53S:R58H:L117T 27,053 Q108K:K40L:F16Y:Y19W:A33H:T51V:T53S:R58H:L117T 28,008 Q108K:K40L:F16Y:Y19W:A33Y:Q38E:T51V:T53S:R58H 36,571 Q108K:K40L:Q4T:F16Y:Y19W:A33H:T51V:T53S:R58H 32,281 Q108K:K40L:F16Y:T51V:T53S:R58H:L117Y 29,375 Q108K:K40L:F16Y:A33H:T51V:T53S:R58H:L117Y 30,800 Q108K:K40L:Y19W:A33H:T51V:T53E:R58H:L117Q 30,670 Q108K:K40L:Y19W:A33H:T51V:T53E:R58H:L117H 32,650 254 Table IV-4 (cont’d) Q108K:K40L:Q38E:T51V:T53S:R58H:L117H 26,340 Q108K:K40L:F16Y:A33H:T51V:T53S:R58H:L117C 27,969 Q108K:K40L:F16Y:Y19W:A33H:T51V:T53S:R58H:L117C 32,803 Q108K:K40L:Q38E:T51V:T53S:R58H:L117C 25,190 Q108K:K40L:Q4S:F16Y:A33H:T51V:T53S:R58H 28,225 Q108K:K40L:Q4Y:Y19W:A33H:T51V:T53E:R58H 28,601 Q108K:K40L:F16Y:A33H:T51V:T53S:R58H:L117S 28,601 Q108K:K40L:F16Y:Y19W:A33H:T51V:T53S:R58H:L117S 31,516 Q108K:K40E:Y19W:A33Y:T51V:T53S:R58H 32,310 Q108K:K40L:F16Y:A33Y:T51V:T53S:R58H 30,012 Q108K:K40L:Y19W:A33H:T51V:T53E:R58H:L117C 31,567 Q108K:K40L:F16Y:T51V:T53S:R58H:L117C 30,063 Q108K:K40E:F16Y:T51V:T53S:R58H 28,681 Q108K:K40L:Y19W:A33Y:T51V:T53S:R58H:L117C 33,809 Q108K:K40L:Y19W:A33H:T51V:T53S:R58H:L117C 28,398 Q108K:K40L:A33H:T51V:T53S:R58H:L77H 27,535 Q108K:K40L:A33H:T51V:T53S:R58H:L77Y 28,331 Q108K:K40L:T51V:T53S:R58H:F16Y:L77Y 27,506 Q108K:K40L:F16Y:Y19W:A33H:T51V:T53S:R58H:L117E 32,378 Q108K:K40L:Y19W:A33Y:T51V:T53S:R58Y 36,299 Q108K:K40L:F16Y:T51V:T53S:R58Y 31,297 Q108K:K40L:F16Y:Y19W:A33H:T51V:T53S:R58Y 34,308 255 Table IV-4 (cont’d) Q108K:K40L:F16Y:A33Y:T51V:T53S:R58Y 31,774 Q108K:K40T:F16Y:Y19W:A33H:T51V:T53S:R58H:L117C 33,233 Q108K:K40Y:F16Y:Y19W:A33H:T51V:T53S:R58H:L117C 34,618 Q108K:K40L:F16Y:Y19W:A33H:T51V:T53S:R58H:L117M 31,761 Q108K:K40D:F16Y:Y19W:A33H:T51V:T53S:R58H:L117C 32,171 Q108K:K40Q:F16Y:Y19W:A33H:T51V:T53S:R58H:L117C 33,434 Q108K:K40E:Q4F:F16Y:Q38F:T53S:R58H 29,635 Q108K:K40C:Q4F:F16Y:Y19W:A33H:T51V:T53S:R58H:L117C 32,514 Q108K:K40N:Q4F:F16Y:Y19W:A33H:T51V:T53S:R58H:L117C 32,606 Q108K:K40H:Q4F:F16Y:Y19W:A33H:T51V:T53S:R58H:L117C 31,227 Q108K:K40S:Q4F:F16Y:Y19W:A33H:T51V:T53S:R58H:L117C 33,162 Q108K:K40E:Q4F:F16Y:Q38F:T53A:R58H 24,829 Q108K:K40L:F16Y:Y19W:A33H:T51V:T53S:R58H:L77Y:L117C 34,691 Q108K:K40E:Q4F:A33H:Q38F:T53A:R58H 26,772 Q108K:K40E:Q4F:F16Y:A33H:Q38F:T53A:R58H 30,458 Q108K:K40E:Q4F:F16Y:Q38F:T53A:R58Y 31,173 Q38P 26,828 Q108K:K40E:Q4F:Y19W:Q38F:T53A:R58H 34,783 Q108K:K40E:Q4F:F16Y:Y19W:Q38F:T53A:R58H 40,509 Q108K:K40E:Q4F:T29Y:Q38F:T53A:R58H 29,354 Q108K:K40E:Q4F:F16Y:T29Y:Q38F:T53A:R58H 31,156 Q108K:K40E:Q4F:Q38F:T53A:R58H:L77M 30,509 256 Table IV-4 (cont’d) Q108K:K40E:Q4F:F16Y:Q38F:T53A:R58H:L77M 34,440 Q108K:K40E:Q4F:F16Y:A33M:Q38F:T53A:R58H 30,877 Q108K:K40L:F16Y:Y19W:T29Y:A33H:T51V:T53S:R58H:L117C 34,288 Q108K:K40E:Q4F:F16Y:A33C:Q38F:T53A:R58H 31,598 Q108K:K40E:Q4F:F16Y:Q38F:T51M:T53A:R58H 28,998 Q108K:K40E:Q4F:Q38F:T51M:T53A:R58H 28,519 Q108K:K40L:F16Y:Y19W:A33H:T51M:T53A:R58H:L117C 33,412 Q108K:K40L:F16Y:Y19W:A33H:T51M:T53S:R58H:L117C 34,361 Q108K:K40E:Q4F:A33M:Q38F:T53A:R58H 28,116 Q108K:K40E:Q4F:F16Y:Q38Y:T53A:R58H 31,815 Q108K:K40E:Q4F:F16Y:M20Y:Q38F:T53A:R58H 29,132 Q108K:K40E:Q4F:M20Y:Q38F:T53A:R58H 29,096 Q108K:K40L:F16Y:Y19W:T29Y:A33H:T51C:T53S:R58H:L117C 33,99 Q108K:K40E:Q4F:F16Y:T29Y:Q38Y:T53A:R58H 32,318 Q108K:K40L:F16Y:Y19W:T29Y:A33H:T51V:T53C:R58H:L117C 32,744 Q108K:A28C:L36C:T51D:F57H 28,122 Q108K:K40E:Q4F:A33H:Q38F:T53A:R58H:L77Y 28,955 Q108K:K40D:Q4F:Q38F:T51V:T53A:R58L:V62E 26,357 53,118 Q108K:K40D:Q4F:Q38F:T53A:R58L:V62E 25,820 56,982 Q108K:K40L:Q4F:Q38F:I42E:T53A:R58L 29,326 Q108K:K40D:Q4F:Q38F:T53A:R58L 28,682 56,298 Q108K:K40D:Q4F:Q38F:T53A:R58H 29,197 257 Table IV-4 (cont’d) Q108K:K40H:Q4F:Q38F:T53A:R58H 28,664 Q108K:K40K:Q4F:Q38F:T53A:R58H 28,439 Q108K:K40N:Q4F:Q38F:T53A:R58H 27,224 Q108K:T51D 28,500 Wild Type hCRBPII 33,068 Wild Type CRABPII 19,127 Q108K:K40E:Q4F:F16Y:Q38H:T53A:R58H 30,840 Q108K:K40E:Q4F:F16Y:Q38Y:T53A:R58H:L117E 35,000 Q108K:K40R:Q4F:Q38F:T53A:R58H 26,242 Q108K:K40L:F16Y:Y19W:A33H:T51V:T53C:R58H:L77Y:L117C 35,025 Q108K:K40L:A33H:T51V:T53S:R58H:L77Y:L117C 29,103 Q108K:K40L:F16Y:Y19W:T29Y:T51V:T53C:R58H:L77Y:L117C 33,813 Q108K:K40L:F16Y:Y19W:T29Y:A33H:T51V:T53C:R58H:L77Y:L117C 43,453 Q108K:K40L:Q38E:T51V:T53S:R58L:L117T 27,263 Q108K:K40L:F16Y:Y19W:T29F:A33H:T51V:T53C:R58H:L77Y:L117C 36,463 Q108K:K40L:F16Y:Y19W:A33H:T51V:T53C:R58H:L77F:L117C 36,747 Q108K:K40L:F16Y:Y19W:T29F:A33H:T51V:T53C:R58H:L117C 38,713 Q108K:K40L:F16Y:Y19W:A33H:T51V:T53C:R58H:L77W:L117C 40,083 Q108K:K40L:F16Y:Y19W:T29W:A33H:T51V:T53C:R58H:L117C 38,757 Q108K:K40L:Y19W:A33H:T51V:T53C:R58H:L77Y:L117C 33,205 Q108K:K40L:Q4F:Q38F:T53A:R58H 28,241 Q108K:K40L:Q4F:Q38F:T53A:R58L 31,014 258 Table IV-4 (cont’d) Q108K:K40E:Q4F:Q38F:T53A:R58L:V62E 26,787 Q108K:K40L:Q4F:T29L:A33W:T51V:T53C:R58W:L117E 36,239 Q108A:K40L:Q44K:T51V:T53S 23,406 Q108K:K40L:Q4E:Q38E:T51V:T53S:R58H 25,764 Q108K:K40L:Q4F:Y19W:T51V:T53S:R58W:L117D:L77D 39,341 Q108K:K40L:Y19W:T29R:A33W:T51V:T53S:R58W:L117E 45,051 Q108K:K40L:Q4F:Y19W:T51V:T53S:R58W:S76R:L117D 46,135 T53D 33,678 T53E 26,630 L119D 22,592 R58A 30,005 IV.3.3 pKa measurements of hCRBPII/chromophore complexes For pKa measurement, protein (20 μM in PBS) was incubated with ligand (0.5 equiv) at room temperature (23 °C) until Schiff base (SB) or protonated Schiff base (PSB) formation was complete. This was verified by UV-Vis spectrum. The solution was then titrated with acid (1 M NaOAc, pH=4) for mutants with low pKa or base (1 M NaOH) for mutants with high pKa in ~ 0.5 pH units, and the absorption spectra were recorded at each point. The λmax of the protein/ligand complex versus pH was plotted. a polynomial fit of the data (3rd power) was applied for pKa determination: !A0 !A = + constant (1 + 10 pH - pKa) 259 The two parameters are: ΔA, the total absorbance change of the PSB (or SB for some proteins) during each point of titration in comparison to the starting absorption, and pKa, the midpoint of titration. It should be noted that a constant is included to account for the deviation from zero absorbance intensity of the deprotonated PSB. pH values were recorded with an accumetTM Basic pH meter (Fisher Scientific) equipped with a PerpHectTM ROSSTM Micro Combination pH electrode (Thermo Scientific Orion). IV.3.4 hCRBPII/chromophore binding Kinetic measurements Binding kinetics were measured at 23 °C using a Cary temperature controller. hCRBPII mutant 20 𝜇M in PBS 2x buffer (pH=7.4) was mixed with 0.5 equivalent of the chromophore. The spectra were recorded immediately after mixing the protein and chromophore, and the absorbance intensity was plotted as a function of time. IV.3.4.A. Pseudo-first-order binding rate measurement Proteins with low pKa (pKa < 9) forming both SB and PSB under the kinetic measurement conditions were fit to a pseudo-first-order rate equation, as the chromophore concentration could not be determined accurately. The fit for the pseudo-first-order rate equation is shown in the equation below, A = A0 × ( 1 − e -kt ) + c where A is the absorbance value at each recorded time point, A0 is the final absorbance value (after the completion of the reaction), k is the pseudo-first-order 260 rate constant, t is the time elapsed, and c is a varying constant which accounts for any time delay from the addition of the chromophore to the point recording was started. This equation was rewritten in KaleidaGraph in the following format, where m2 is the rate constant, and the half-life (t1/2) of the reaction can be calculated by ln(2)/k. y = m1 × ( 1 − e -m2 × m0 ) + m3 IV.3.4.B. Second-order binding rate measurement For proteins with high imine pKa (pKa > 9), the majority of the protein complex is in PSB form, and chromophore concentration can be determined confidently. The product concentration can be calculated at each point by Beer’s Law using absorbance intensity at each time point and the extinction coefficient of the hCRBPII/ligand complex. Then, the concentration of product (hCRBPII/ligand complex) versus time was plotted. The data were fit to a second-order rate equation as shown in the equation below, which was derived for product formation following the second-order rate equation previously.4,5 1 y = m3 − ( m2 × m0 ) + m1 where m3 is the concentration of the limiting reactant (in all cases, this should be 10 μM, assuming greater than 50% of the protein is functional), and m2 is the rate constant k. The half-life (t1/2) of the reaction can be calculated by 1/(k*[A]0), wherein 10 μM was assumed for [A]0.6 261 IV.3.5. Absolute fluorescent quantum yield measurements Absolute fluorescence quantum yields (Φ) were recorded at room temperature on a Quantaurus-QY C11347-11 (Hamamatsu Photonics K.K., Japan) equipped with a Xenon lamp and a monochromator as excitation light source, an integration sphere, and a multichannel back-thinned CCD detector. All samples were diluted with PBS.2X solution or corresponding organic solvents (A < 0.1). Recorded values are average numbers (n = 5). IV.4 Cloning to mammalian expression vectors IV.4.1 General cloning protocol The DNA fragment was amplified using Pfu Turbo DNA Polymerase (Agilent) with the appropriate primers depending on the restriction site to be introduced to the template plasmid. All restriction sites for cloning were chosen with sticky ends. PCR conditions are specified in Table IV-5 using a Bio-Rad iCycler thermal cycler. The PCR amplified DNA fragment was purified by Wizard® SV Gel and PCR Clean-Up System (Promega) from 1% agarose gel in an amount of 20-50 ng/μL. The fragment was digested with proper enzymes and ligated to a similarly prepared plasmid (50 ng/μL). Ligation between insert fragment and plasmid was performed with 30 ng of plasmid and 90 ng of insert using T4 DNA Ligase (New England BioLabs). The ligated product was transformed into E. coli XL-1 blue competent cells and grown on LB-agar plates supplemented with antibiotics (100 μg/mL ampicillin, 7.5 μg/mL tetracycline) at 37 °C for 20 hours. Colonies (3-6) were 262 inoculated in LB medium (10 mL) containing the proper amount of antibiotics (100 μg/mL ampicillin, 7.5 μg/mL tetracycline) and incubated at 37 °C while shaking, for 20 hours. Table IV-5. PCR cycling conditions for cloning. PCR Program Time (min) 1x 97 °C 1:00 1x 95 °C 3:00 95 °C 00:30 72°C 00:45 15x 72 °C (temp decreases after cycle 1 by 1 °C by every 03:40 cycle) 95 °C 00:30 20x 3-5 °C Below Primer Tm 00:45 72 °C 03:40 1x 72 °C 08:00 1x 4 °C 10:00 Reactant volume DNA (Template Plasmid) 100 ng (x μl) Primer forward 50 pmol (y μl) Primer reverse 50 pmol (z μl) 10 mM dNTP 1 μL DMSO 1 μL 50 mM MgCl2 1 μL 5× Pfu buffer 5 μL Pfu Turbo DNA Polymerase 1.5 μL DI Water 50 – x – y – z – 9.5 μL 263 DNA purification was performed using the Promega Wizard® Plus SV Miniprep DNA purification kit (A1330) following the suggested protocol. The DNA sequence was verified with the corresponding sequencing primers by the MSU gene sequencing facility. Sequencing primers used are shown below. CMV end_Seq: 5’-GGT CTA TAT AAG CAG AGC TGG TTT AG-3’ midGFP: 5’-CGT GCT GCT GCC CGA CAA CC-3’ IV.4.2 Sequences of plasmids described in this thesis Plasmid 1: HindIII-EGFP-NotI- Q108K:K40L:F16Y:Y19W:T29Y:A33H:T51V:T53C:R58H:L117C-EcoRI-3NLS- Stop-BamHI (pFlag-CMV2) AAGCTTATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGA GCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATG CCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCC TGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGA CCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGC GCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAG GGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAA CATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCG ACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGC AGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCT GCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGA AGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATG GACGAGCTGTACAAGTCCGGAGCCGCTGCAGGAGGCGGCCGCATGACGAGGGACCAGAA TGGAACCTGGGAGATGGAGAGTAATGAAAACTATGAGGGCTGGATGAAGGCCCTGGATA TTGATTTTGCCTACCGCAAGATTCACGTACGTCTCACTCAGACGCTGGTTATTGATCAA GATGGTGATAACTTCAAGGTAAAATGCACTAGCACATTCCACAACTATGATGTGGATTT CACTGTTGGAGTAGAGTTTGACGAGTACACAAAGAGCCTGGATAACCGGCATGTTAAGG CACTGGTCACCTGGGAAGGTGATGTCCTTGTGTGTGTGCAAAAGGGGGAGAAGGAGAAC CGCGGCTGGAAGAAGTGGATTGAGGGGGACAAGCTGTACTGTGAGCTGACCTGTGGTGA CCAGGTGTGCCGTCAAGTGTTCAAAAAGAAGTGCGAATTCGCTGACCCCAAGAAGAAGA GGAAGGTGGACCCCAAGAAGAAGAGGAAGGTGGACCCCAAGAAGAAGAGGAAGGTGTGA AAACATCGATAGATCTGATATC 264 Plasmid 2: HindIII-EGFP-NotI- Q108K:K40L:F16Y:Y19W:T29Y:A33H:T51V:T53C:R58H:L117C-EcoRI-NES- Stop-BamHI (pFlag-CMV2) AAGCTTATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGA GCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATG CCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCC TGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGA CCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGC GCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAG GGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAA CATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCG ACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGC AGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCT GCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGA AGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATG GACGAGCTGTACAAGCCGGAGCCGCTGCAGGAGGCGGCCGCATGACGAGGGACCAGAAT GGAACCTGGGAGATGGAGAGTAATGAAAACTATGAGGGCTGGATGAAGGCCCTGGATAT TGATTTTGCCTACCGCAAGATTCACGTACGTCTCACTCAGACGCTGGTTATTGATCAAG ATGGTGATAACTTCAAGGTAAAATGCACTAGCACATTCCACAACTATGATGTGGATTTC ACTGTTGGAGTAGAGTTTGACGAGTACACAAAGAGCCTGGATAACCGGCATGTTAAGGC ACTGGTCACCTGGGAAGGTGATGTCCTTGTGTGTGTGCAAAAGGGGGAGAAGGAGAACC GCGGCTGGAAGAAGTGGATTGAGGGGGACAAGCTGTACTGTGAGCTGACCTGTGGTGAC CAGGTGTGCCGTCAAGTGTTCAAAAAGAAGTGCGAATTCGAGCTTGCCGAGAAACTTGC CGGGCTTGACATAAATTGAGGATCCCGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCT Plasmid 3: HindIII-EGFP-NotI- Q108K:K40L:F16Y:Y19W:T29Y:A33H:T51V:T53C:R58H:L117C-EcoRI-Linker (whole cell localization)-Stop-BamHI (pFlag-CMV2) AAGCTTATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGA GCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATG CCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCC TGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGA CCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGC GCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAG GGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAA CATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCG ACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGC AGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCT GCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGA 265 AGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATG GACGAGCTGTACAAGGGCGGCCGCATGACGAGGGACCAGAATGGAACCTGGGAGATGGA GAGTAATGAAAACTATGAGGGCTGGATGAAGGCCCTGGATATTGATTTTGCCTACCGCA AGATTCACGTACGTCTCACTCAGACGCTGGTTATTGATCAAGATGGTGATAACTTCAAG GTAAAATGCACTAGCACATTCCACAACTATGATGTGGATTTCACTGTTGGAGTAGAGTT TGACGAGTACACAAAGAGCCTGGATAACCGGCATGTTAAGGCACTGGTCACCTGGGAAG GTGATGTCCTTGTGTGTGTGCAAAAGGGGGAGAAGGAGAACCGCGGCTGGAAGAAGTGG ATTGAGGGGGACAAGCTGTACTGTGAGCTGACCTGTGGTGACCAGGTGTGCCGTCAAGT GTTCAAAAAGAAGTGCGAATTCATCGATAGATCTGATATCGGTACCAGTCGACTCTAGA GGATCCCGGGTGGCATCCCTGTGACCCCTCCCCA Plasmid 4: HindIII-EGFP-NotI-Q108K:K40E:Q4F:Q38F:T53A:R58H-EcoRI- 3NLS-Stop-BamHI (pFlag-CMV2) AAGCTTATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGA GCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATG CCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCC TGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGA CCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGC GCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAG GGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAA CATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCG ACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGC AGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCT GCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGA AGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATG GACGAGCTGTACAAGGGCGGCCGCATGACGAGGGACTTCAATGGAACCTGGGAGATGGA GAGTAATGAAAACTTTGAGGGCTACATGAAGGCCCTGGATATTGATTTTGCCACCCGCA AGATTGCAGTACGTCTCACTTTTACGGAAGTTATTGATCAAGATGGTGATAACTTCAAG ACAAAAGCCACTAGCACATTCCACAACTATGATGTGGATTTCACTGTTGGAGTAGAGTT TGACGAGTACACAAAGAGCCTGGATAACCGGCATGTTAAGGCACTGGTCACCTGGGAAG GTGATGTCCTTGTGTGTGTGCAAAAGGGGGAGAAGGAGAACCGCGGCTGGAAGAAGTGG ATTGAGGGGGACAAGCTGTACCTGGAGCTGACCTGTGGTGACCAGGTGTGCCGTCAAGT GTTCAAAAAGAAGTGCGAATTCGCTGACCCCAAGAAGAAGAGGAAGGTGGACCCCAAGA AGAAGAGGAAGGTGGACCCCAAGAAGAAGAGGAAGGTGTGAAAACATCGATAGATCTGA Plasmid 5: HindIII-EGFP-NotI- Q108K:K40E:Q4F:Q38F:T53A:R58H-EcoRI- NES-Stop-BamHI (pFlag-CMV2) AAGCTTATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGA GCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATG CCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCC 266 TGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGA CCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGC GCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAG GGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAA CATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCG ACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGC AGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCT GCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGA AGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATG GACGAGCTGTACAAGGGCGGCCGCATGACGAGGGACTTCAATGGAACCTGGGAGATGGA GAGTAATGAAAACTTTGAGGGCTACATGAAGGCCCTGGATATTGATTTTGCCACCCGCA AGATTGCAGTACGTCTCACTTTTACGGAAGTTATTGATCAAGATGGTGATAACTTCAAG ACAAAAGCCACTAGCACATTCCACAACTATGATGTGGATTTCACTGTTGGAGTAGAGTT TGACGAGTACACAAAGAGCCTGGATAACCGGCATGTTAAGGCACTGGTCACCTGGGAAG GTGATGTCCTTGTGTGTGTGCAAAAGGGGGAGAAGGAGAACCGCGGCTGGAAGAAGTGG ATTGAGGGGGACAAGCTGTACCTGGAGCTGACCTGTGGTGACCAGGTGTGCCGTCAAGT GTTCAAAAAGAAGTGCGAATTCGAGCTTGCCGAGAAACTTGCCGGGCTTGACATAAATT GAGGATCCCGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCT Plasmid 6: HindIII-EGFP-NotI-Q108K:K40E:Q4F:Q38F:T53A:R58H-EcoRI- Linker (whole cell localization)-Stop-BamHI (pFlag-CMV2) AAGCTTATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGA GCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATG CCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCC TGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGA CCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGC GCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAG GGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAA CATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCG ACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGC AGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCT GCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGA AGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATG GACGAGCTGTACAAGGGCGGCCGCATGACGAGGGACTTCAATGGAACCTGGGAGATGGA GAGTAATGAAAACTTTGAGGGCTACATGAAGGCCCTGGATATTGATTTTGCCACCCGCA AGATTGCAGTACGTCTCACTTTTACGGAAGTTATTGATCAAGATGGTGATAACTTCAAG ACAAAAGCCACTAGCACATTCCACAACTATGATGTGGATTTCACTGTTGGAGTAGAGTT TGACGAGTACACAAAGAGCCTGGATAACCGGCATGTTAAGGCACTGGTCACCTGGGAAG GTGATGTCCTTGTGTGTGTGCAAAAGGGGGAGAAGGAGAACCGCGGCTGGAAGAAGTGG ATTGAGGGGGACAAGCTGTACCTGGAGCTGACCTGTGGTGACCAGGTGTGCCGTCAAGT GTTCAAAAAGAAGTGCGAATTCATCGATAGATCTGATATCGGTACCAGTCGACTCTAGA GGATCCCGGGTGGCATCCCTGTGACCCCTCCCC 267 Plasmid 7: HindIII-EGFP-NotI-Q108K:K40E:Q4F:Q38F:T53A:R58L-EcoRI- 3NLS-Stop-BamHI (pFlag-CMV2) AAGCTTATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGA GCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATG CCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCC TGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGA CCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGC GCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAG GGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAA CATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCG ACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGC AGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCT GCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGA AGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATG GACGAGCTGTACAAGTCCGGAGCCGCTGCAGGAGGCGGCCGCATGACGAGGGACTTCAA TGGAACCTGGGAGATGGAGAGTAATGAAAACTTTGAGGGCTACATGAAGGCCCTGGATA TTGATTTTGCCACCCGCAAGATTGCAGTACGTCTCACTTTTACGGAAGTTATTGATCAA GATGGTGATAACTTCAAGACAAAAGCCACTAGCACATTCCTGAACTATGATGTGGATTT CACTGTTGGAGTAGAGTTTGACGAGTACACAAAGAGCCTGGATAACCGGCATGTTAAGG CACTGGTCACCTGGGAAGGTGATGTCCTTGTGTGTGTGCAAAAGGGGGAGAAGGAGAAC CGCGGCTGGAAGAAGTGGATTGAGGGGGACAAGCTGTACCTGGAGCTGACCTGTGGTGA CCAGGTGTGCCGTCAAGTGTTCAAAAAGAAGTGCGAATTCGCTGACCCCAAGAAGAAGA GGAAGGTGGACCCCAAGAAGAAGAGGAAGGTGGACCCCAAGAAGAAGAGGAAGGTGTGA AAACATCGATAGATCTGATATCGGTACCAGTCGA Plasmid 8: HindIII-EGFP-NotI-Q108K:K40E:Q4F:Q38F:T53A:R58L-EcoRI- NES-Stop-BamHI (pFlag-CMV2) AAGCTTATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGA GCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATG CCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCC TGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGA CCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGC GCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAG GGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAA CATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCG ACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGC AGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCT GCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGA AGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATG GACGAGCTGTACAAGTCCGGAGCCGCTGCAGGAGGCGGCCGCATGACGAGGGACTTCAA TGGAACCTGGGAGATGGAGAGTAATGAAAACTTTGAGGGCTACATGAAGGCCCTGGATA 268 TTGATTTTGCCACCCGCAAGATTGCAGTACGTCTCACTTTTACGGAAGTTATTGATCAA GATGGTGATAACTTCAAGACAAAAGCCACTAGCACATTCCTGAACTATGATGTGGATTT CACTGTTGGAGTAGAGTTTGACGAGTACACAAAGAGCCTGGATAACCGGCATGTTAAGG CACTGGTCACCTGGGAAGGTGATGTCCTTGTGTGTGTGCAAAAGGGGGAGAAGGAGAAC CGCGGCTGGAAGAAGTGGATTGAGGGGGACAAGCTGTACCTGGAGCTGACCTGTGGTGA CCAGGTGTGCCGTCAAGTGTTCAAAAAGAAGTGCGAATTCGAGCTTGCCGAGAAACTTG CCGGGCTTGACATAAATTGAGGATCCCGGGTGGCATCCCTGTGACCCCTCCCCAGTGCC Plasmid 9: HindIII-EGFP-NotI-Q108K:K40E:Q4F:Q38F:T53A:R58L-EcoRI- Linker (whole cell localization)-Stop-BamHI (pFlag-CMV2) AAGCTTATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGA GCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATG CCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCC TGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGA CCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGC GCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAG GGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAA CATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCG ACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGC AGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCT GCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGA AGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATG GACGAGCTGTACAAGGGCGGCCGCATGACGAGGGACCAGAATGGAACCTGGGAGATGGA GAGTAATGAAAACTATGAGGGCTGGATGAAGGCCCTGGATATTGATTTTGCCTACCGCA AGATTCACGTACGTCTCACTCAGACGCTGGTTATTGATCAAGATGGTGATAACTTCAAG GTAAAATGCACTAGCACATTCCACAACTATGATGTGGATTTCACTGTTGGAGTAGAGTT TGACGAGTACACAAAGAGCCTGGATAACCGGCATGTTAAGGCACTGGTCACCTGGGAAG GTGATGTCCTTGTGTGTGTGCAAAAGGGGGAGAAGGAGAACCGCGGCTGGAAGAAGTGG ATTGAGGGGGACAAGCTGTACTGTGAGCTGACCTGTGGTGACCAGGTGTGCCGTCAAGT GTTCAAAAAGAAGTGCGAATTCATCGATAGATCTGATATCGGTACCAGTCGACTCTAGA GGATCCCGGGTGGCATCCCTGTGACC Plasmid 10: HindIII-EGFP-NotI- Q108K:K40L:F16Y:Y19W:A33H:T51V:T53C:R58H:L77Y:L117C-EcoRI-3NLS- Stop-BamHI (pFlag-CMV2) AAGCTTATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGA GCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATG CCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCC TGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGA CCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGC 269 GCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAG GGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAA CATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCG ACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGC AGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCT GCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGA AGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATG GACGAGCTGTACAAGTCCGGAGCCGCTGCAGGAGGCGGCCGCATGACGAGGGACCAGAA TGGAACCTGGGAGATGGAGAGTAATGAAAACTATGAGGGCTGGATGAAGGCCCTGGATA TTGATTTTGCCACCCGCAAGATTCACGTACGTCTCACTCAGACGCTGGTTATTGATCAA GATGGTGATAACTTCAAGGTAAAATGCACTAGCACATTCCACAACTATGATGTGGATTT CACTGTTGGAGTAGAGTTTGACGAGTACACAAAGAGCTACGATAACCGGCATGTTAAGG CACTGGTCACCTGGGAAGGTGATGTCCTTGTGTGTGTGCAAAAGGGGGAGAAGGAGAAC CGCGGCTGGAAGAAGTGGATTGAGGGGGACAAGCTGTACTGTGAGCTGACCTGTGGTGA CCAGGTGTGCCGTCAAGTGTTCAAAAAGAAGTGCGAATTCGCTGACCCCAAGAAGAAGA GGAAGGTGGACCCCAAGAAGAAGAGGAAGGTGGACCCCAAGAAGAAGAGGAAGGTGTGA AAACATCGATAGATCTGATATCGGTACCAGTCGACTCTAGAGGATC Plasmid 11: HindIII-EGFP-NotI- Q108K:K40L:F16Y:Y19W:A33H:T51V:T53C:R58H:L77Y:L117C-EcoRI-NES- Stop-BamHI (pFlag-CMV2) AAGCTTATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGA GCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATG CCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCC TGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGA CCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGC GCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAG GGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAA CATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCG ACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGC AGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCT GCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGA AGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATG GACGAGCTGTACAAGTCCGGAGCCGCTGCAGGAGGCGGCCGCATGACGAGGGACCAGAA TGGAACCTGGGAGATGGAGAGTAATGAAAACTATGAGGGCTGGATGAAGGCCCTGGATA TTGATTTTGCCACCCGCAAGATTCACGTACGTCTCACTCAGACGCTGGTTATTGATCAA GATGGTGATAACTTCAAGGTAAAATGCACTAGCACATTCCACAACTATGATGTGGATTT CACTGTTGGAGTAGAGTTTGACGAGTACACAAAGAGCTACGATAACCGGCATGTTAAGG CACTGGTCACCTGGGAAGGTGATGTCCTTGTGTGTGTGCAAAAGGGGGAGAAGGAGAAC CGCGGCTGGAAGAAGTGGATTGAGGGGGACAAGCTGTACTGTGAGCTGACCTGTGGTGA CCAGGTGTGCCGTCAAGTGTTCAAAAAGAAGTGCGAATTCGAGCTTGCCGAGAAACTTG CCGGGCTTGACATAAATTGAGGATCCCGGGTGGCATCCCTGTGACCCCTCCCCAGTGCC 270 Plasmid 12: HindIII-EGFP-NotI- Q108K:K40L:F16Y:Y19W:A33H:T51V:T53C:R58H:L77Y:L117C-EcoRI-Linker (whole cell localization)-Stop-BamHI (pFlag-CMV2) AAGCTTATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGA GCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATG CCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCC TGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGA CCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGC GCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAG GGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAA CATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCG ACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGC AGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCT GCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGA AGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATG GACGAGCTGTACAAGGGCGGCCGCATGACGAGGGACCAGAATGGAACCTGGGAGATGGA GAGTAATGAAAACTATGAGGGCTGGATGAAGGCCCTGGATATTGATTTTGCCACCCGCA AGATTCACGTACGTCTCACTCAGACGCTGGTTATTGATCAAGATGGTGATAACTTCAAG GTAAAATGCACTAGCACATTCCACAACTATGATGTGGATTTCACTGTTGGAGTAGAGTT TGACGAGTACACAAAGAGCTACGATAACCGGCATGTTAAGGCACTGGTCACCTGGGAAG GTGATGTCCTTGTGTGTGTGCAAAAGGGGGAGAAGGAGAACCGCGGCTGGAAGAAGTGG ATTGAGGGGGACAAGCTGTACTGTGAGCTGACCTGTGGTGACCAGGTGTGCCGTCAAGT GTTCAAAAAGAAGTGCGAATTCATCGATAGATCTGATATCGGTACCAGTCGACTCTAGA GGATCCCGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGT IV.5 Mammalian cell culture and transfection All cell lines (HeLa and COS-7) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, containing phenol red, 4.5 g/L D-glucose, L-glutamine, and 110 mg/L Sodium Pyruvate; purchased from Sigma-Aldrich) supplemented with 10% (v/v) Fetal Bovine Serum (FBS, BioWest) and 1x Penicillin-Streptomycin- Glutamine (PSG; purchased from GIBCO) at 37 °C within a 5% CO2 and 10% O2 atmosphere. 271 Cells were seeded 2 d before transfection on an ibidi μ-Slide 8 well coverslip (with ibiTreat). Transient transfection was performed at ~70% confluency with 0.25 μg of plasmid DNA (per well) using Genjet Ver. II (purchased from SignaGen) according to the manufacturer’s protocol. The media was replaced with complete serum/antibiotics containing medium after 5 h. After about another 20 h incubation, cells were washed twice with Dulbecco’s phosphate-buffered saline (DPBS, supplemented with calcium chloride and magnesium chloride; Sigma-Aldrich) and incubated in Phenol red-free RPMI 1640 medium (Sigma-Aldrich). Prior to confocal imaging, the fluorophore (in DMSO solution) was diluted to the specified concentration noted in text with pre-heated (37 °C) media and was added to the cells. For mutants that need more time to form the complex, the chromophore was added 2 h or the night before imaging. All washing steps were omitted. IV.6 General methods for confocal imaging Confocal images were acquired using an inverted laser scanning confocal microscope; Olympus FluoView 1000 spectral-based laser scanning confocal microscope configured on an Olympus IX81 automated inverted microscope platform, equipped with blue diode laser (405 nm), the Argon gas laser (458 nm, 488 nm, and 514 nm), the green diode laser (559 nm) and the red Helium-Neon gas laser (635 nm) sources. Differential Interference Contrast (DIC) images were acquired using a μ-Slide DIC lid (ibidi). All images were taken with Olympus UPIanFL N 40x/1.30 oil and PlanApo N 60x/1.40 oil objectives, and Olympus Fluoview version 4.2a software was used to process images. 272 hCRBPII/ThioPhenol and hCRBPII/CyThioPhenol were imaged using multiple settings, as indicated in the main text. 1) For the hydroxyl moiety single ESPT of ThioPhenol: 515 nm excitation, excitation dichroic mirrors (DM) 405- 440/515 nm and bandpass 620 nm-720 nm. 2) For the hydroxyl moiety single ESPT of CyThioPhenol: 559 nm excitation, excitation dichroic mirrors (DM) 405/488/559 and bandpass 620 nm-720 nm. 3) For the double ESPT of ThioPhenol: 405 nm excitation, excitation dichroic mirrors (DM) 405/488 nm and bandpass 560 nm-660 nm. 4) For the imine bond single ESPT of CyThioPhenol: 405 nm excitation, excitation dichroic mirrors (DM) 405/488 nm, and bandpass 480 nm-580 nm. 5) For the green fluorescence of EGFP: 488 nm excitation, excitation dichroic mirrors (DM) 405/488 nm and bandpass 500 nm-550 nm. DIC images were acquired using a μ-Slide DIC lid (ibidi). Kalman averaging was applied each time prior to each imaging experiment. Fluorescence in each experiment was normalized to the same intensity, including the same laser intensity, gain, and amplifier offset. All images are pseudocolored with green, red, or cyan colors. The following section describes the synthesis procedures of several chromophores discussed in chapters II and III, synthesized and purified by Mr. Mehdi Moemeni. 273 IV.7 General synthetic procedures Commercially available starting materials were obtained from Sigma- Aldrich and were used without further purification unless specified. All moisture sensitive reactions were carried out in flame-dried or oven-dried glassware under an atmosphere of nitrogen or argon. Unless otherwise mentioned, solvents were purified as follows: tetrahydrofuran (THF) and diethyl ether (Et2O) were distilled freshly from the classical sodium/benzophenone ketyl still pot; dichloromethane (DCM), acetonitrile, and toluene were dried over CaH2 and freshly distilled prior to use; dimethylsulfoxide (DMSO), dimethylformamide (DMF), and triethylamine (Et3N) were distilled from CaH2 and stored over activated molecular sieves. Chemical shifts were reported relative to the residual solvent peaks. (1H-NMR: 𝛿 7.26 ppm for CDCl3, 𝛿 3.31 ppm for CD3OD, 𝛿 2.50 ppm for DMSO-d6, 2.05 ppm for Aceton-d6 respectively. 13C-NMR: 𝛿 77.16 ppm for CDCl3, 𝛿 49.00 ppm for CD3OD, 𝛿 39.52 ppm for DMSO-d6, 206.68 and 29.92 ppm for Aceton-d6 respectively.) Analytical thin layer chromatography (TLC) was performed with pre- coated silica gel 60 F254 plates (Analtech, Inc.) Compounds in TLC were visualized upon UV irradiation and various staining techniques, i.e., p-anisaldehyde, potassium permanganate, phosphomolybdic acid in ethanol. Silica gel flash column chromatography was performed with Silicycle 40-60 Å (30 ~ 75 μM) silica gel. 274 IV.7.1 Synthesis of Thiophenol O O EtO P O EtO OEt 1.5 equiv. OEt DIBALH, 2.5 equiv. Br S O NaH, 1.5 equiv. S DCM, -78°C, 15 min Br 1 THF, 0°C to reflux, 2 24 h, 61% OH HO B OH O OH DMP, 1.2 equiv. 1.2 equiv. NaHCO3, 1.5 equiv. Pd(PPh3)4, 5 mol% S DCM, 0°C, 30 min Br S Br K2CO3, 10 equiv. 3 two steps 54% 4 toluene/ EtOH/ H2O, (20:7:2), 80°C, 1 h, 51% O S HO ThioPhenol ethyl (E)-3-(5-bromothiophen-2-yl)but-2-enoate (2) Triethyl phosphonoacetate (1.98 mL, 10 mmol) was added dropwise under nitrogen to a suspension of NaH (60% on mineral oil, 400mg, 10 mmol) in dry THF (5mL) at 0 °C and the resulting mixture was stirred for another 30 min at the same temperature. A solution of 2-acetyl-5-bromothiophene 1 (1.35 g, 6.6 mmol) in THF (5 mL) was added dropwise to the mixture and heated to reflux for 24 h. After cooling, the reaction was quenched with saturated NH4Cl and extracted three times with ethyl acetate which dried on Na2SO4. After evaporating the solvent, the product was purified via column chromatography by using hexane/ ethyl acetate (97:3) as eluent to give compound 2 as a yellow solid (1.1 g, 61%). 275 1H NMR (500 MHz, Chloroform-d) 𝛿 7.07 (d, J = 4.0 Hz, 1H), 7.01 (d, J = 4.0 Hz, 1H), 6.13 (q, J = 1.2 Hz, 1H), 4.21 (q, J = 7.2 Hz, 2H), 2.55 (d, J = 1.2 Hz, 3H), 1.32 (t, J = 7.1 Hz, 3H). 13C NMR (126 MHz, Chloroform-d) 𝛿 166.51, 146.77, 146.71, 130.88, 126.96, 114.65, 114.41, 60.03, 16.69, 14.33. (E)-3-(5-bromothiophen-2-yl)but-2-en-1-ol (3) To the compound 2 (819 mg, 3mmol) in dry DCM (10 mL) at -78 °C was added DIBALH (1M in hexane, 7 mL, 7 mmol) dropwise. After 15 min, the reaction was completed, and methanol (5 mL) was added dropwise to quench the reaction. saturated solution Potassium sodium tartrate (15mL) was added with DCM (10 mL). The mixture was stirred for 2 h and extracted with DCM (3 × 10 mL) and the combined organic layers were dried over Na2SO4. Evaporation of solvent gave compound 3 as a yellow solid and it used in next step without further purification. (E)-3-(5-bromothiophen-2-yl)but-2-enal (4) To compound 3 (559 mg, 2.4 mmol) in dry DCM (10 mL) at 0 °C, Dess- Martin periodinane (DMP) (1.22 g, 2.88 mmol) and NaHCO3 (302 mg, 3.6 mmol) was added. Reaction was stirred at this temperature for 30 min and was quenched by addition of saturated sodium thiosulfate (10 mL) and saturated sodium bicarbonate (10 mL). The mixture was extracted with DCM (3 × 10 mL) and the combined organic layers were dried over Na2SO4. After evaporating the solvent, it 276 was purified via column chromatography by using hexane/ ethyl acetate (96:4) as eluent to give compound 4 as a yellow solid (374mg, two steps 54%). 1H NMR (500 MHz, DMSO-d6) 𝛿 10.05 (d, J = 7.6 Hz, 1H), 7.52 (d, J = 4.0 Hz, 1H), 7.33 (d, J = 4.1 Hz, 1H), 6.23 (dq, J = 7.7, 1.2 Hz, 1H), 2.51 (s, 3H). 13C NMR (126 MHz, Chloroform-d) 𝛿 190.47, 148.71, 145.67, 131.37, 128.21, 124.44, 116.73, 15.56. ThioPhenol To the mixture of compound 4 (230 mg, 1.0 mmol), Pd(PPh3)4 (58 mg, 0.05 mmol), and toluene (20.0 mL), a suspension of (4-hydroxyphenyl)boronic acid (165.6 mg, 1.20 mmol) in ethanol (7 mL) and a solution of K2CO3 (1.38 g, 10 mmol) in water (2.0 mL) were added. The mixture was heated to 80 °C in an argon atmosphere for one hour. After cooling to room temperature, the mixture was filtered through Celite, and the organic phase was washed with water. The organic layers were then dried over Na2SO4 and concentrated under vacuum. Column chromatography with hexane/ ethyl acetate (9:1) of the residue afforded a mixture of trans/cis (84:16) isomers of ThioPhenol that were subjected to recrystallization with hexane/ ethyl acetate (7:3) to give pure trans-ThioPhenol as orange crystals (124 mg, 51%). 1H NMR (500 MHz, Chloroform-d) 𝛿 10.042 (d, J= 7.5, 1H), 9.844 (s, 1H), 7.637 (d, J= 4, 1H), 7.534 (d, J= 8.5, 2H), 7.402 (d, J= 4, 1H), 6.812 (d, J= 9, 2H), 6.264 (d, J= 7.5, 1H), 2.542 (s, 3H). 277 13C NMR (126 MHz, Chloroform-d) 𝛿 191.643, 158.669, 150.549, 148.104, 141.318, 131.303, 127.682, 124.403, 123.857, 123.338, 116.442, 15.906. IV.7.2 Synthesis of ThioPhenol-CF3 CF3 OH HO B O O OH 1.2 equiv. S S Pd(PPh3)4, 5 mol% Br K2CO3, 10 equiv. 4 CF3 toluene/ EtOH/ H2O, HO (20:7:2), 80°C, 1 h, 38% ThioPhenol-CF3 The synthesis of ThioPhenol-CF3 was performed according to the general procedure described above (with same scale). Pure ThioPhenol-CF3 was obtained as orange crystals (118 mg, 38%). 1H NMR (500 MHz, Chloroform-d) 𝛿 10.12 (d, J = 7.9 Hz, 1H), 7.41 (d, J = 3.9 Hz, 1H), 7.36 (d, J = 8.5 Hz, 1H), 7.26 (d, J = 3.9 Hz, 1H), 7.09 – 7.04 (m, 2H), 6.47 (dq, J = 7.9, 1.2 Hz, 1H), 2.61 (d, J = 1.1 Hz, 3H). 13C NMR (126 MHz, Chloroform-d) 𝛿 191.26, 156.28, 150.87, 144.33, 143.92, 134.41, 130.41, 130.26, 130.17, 129.05, 128.38, 124.49, 124.03, 122.30, 120.47, 119.05, 118.36, 114.02, 15.82. 278 IV.7.3 Synthesis of ThioPhenol-OMe2 O OH HO B O O OH 1.2 equiv. S S Pd(PPh3)4, 5 mol% Br K2CO3, 10 equiv. 4 HO toluene/ EtOH/ H2O, (20:7:2), O 80°C, 1 h, 41% ThioPhenol-OMe The synthesis of ThioPhenol-OMe2 was performed according to the general procedure described above (with same scale). Pure ThioPhenol-OMe2 was obtained as orange crystals (112 mg, 41%). 1H NMR (500 MHz, Chloroform-d) 𝛿 10.13 (d, J = 7.8 Hz, 1H), 7.41 (d, J = 3.9 Hz, 1H), 7.21 – 7.17 (m, 2H), 7.09 (d, J = 2.0 Hz, 1H), 6.96 (d, J = 8.3 Hz, 1H), 6.45 (dq, J = 7.9, 1.1 Hz, 1H), 5.74 (s, 1H), 3.98 (s, 3H), 2.59 (d, J = 1.1 Hz, 3H). 13C NMR (126 MHz, Chloroform-d) 𝛿 190.52, 149.78, 148.42, 146.80, 146.39, 142.22, 129.25, 126.01, 123.75, 123.23, 119.61, 114.94, 108.52, 56.04, 15.72. 279 IV.7.4 Synthesis of MR0 CH3I, 3 equiv. Br Br KOH, 4 equiv. Br NaOMe, 1.5 equiv. Br KI, 10 mol% CuI, 1 equiv. DMSO, r.t., 6 DMF, reflux, 5 24 h, 88% 1.5 h, 39% n-BuLi, 2 equiv. DMF, 2 equiv. BBr3, 2.5 equiv. O Br O THF/ Hexane, O DCM, 0°C to r.t., -78°C to r.t., 4 h, 31% 7 8 4 h, 63% HO O MR0 2,7-dibromo-9,9-dimethyl-9H-fluorene (6) 2,7-Dibromo-9H-fluorene 5 (7.77 g, 24 mmol) was added to a stirring solution of potassium hydroxide (5.38 g, 96 mmol) and potassium iodide (0.4 g, 2.4 mmol) in dimethylsulforxide (40 mL). Then Iodomethane (4.48 mL, 72 mmol) was added dropwise, and the mixture was stirred at room temperature for 24 hours. After completion, the reaction was poured into 250 mL water and extracted with DCM three times and the combined organic layers were dried over Na2SO4. After evaporating the solvent, the product was purified via flash column chromatography by using DCM as eluent to give compound 6 (7.43 g, 88%). 1H NMR (500 MHz, Chloroform-d) 𝛿 7.57 – 7.52 (m, 4H), 7.46 (dd, J = 8.1, 1.7 Hz, 2H), 1.47 (s, 6H). 280 13C NMR (126 MHz, Chloroform-d) 𝛿 155.25, 137.15, 130.32, 126.20, 121.47, 121.45, 47.32, 26.86. 2-bromo-7-methoxy-9,9-dimethyl-9H-fluorene (7) Sodium methoxide was prepared by adding sodium (96.5 mg, 4.2 mmol) into 1.5 mL of anhydrous methanol under nitrogen. When the sodium disappeared, compound 6 (1 g, 2.8 mmol) in 15 mL of dry DMF and copper iodide (532 mg, 2.8 mmol) was added to the above solution and heated to reflux for 1.5 hour. The reaction mixture was poured into ice water and then extracted with ethyl acetate (3 × 15 mL). The combined organic layers were washed with 1 M HCl and brine and dried over Na2SO4. The resulting crude was purified by column chromatography (hexane/ ethyl acetate 98:2) to give compound 7 as a white solid (330 mg, 39%). 1H NMR (500 MHz, Chloroform-d) 𝛿 7.60 (d, J = 8.3 Hz, 1H), 7.51 (d, J = 1.8 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.43 (dd, J = 8.0, 1.8 Hz, 1H), 6.95 (d, J = 2.4 Hz, 1H), 6.89 (dd, J = 8.3, 2.4 Hz, 1H), 3.88 (s, 3H), 1.46 (s, 6H). 13C NMR (126 MHz, Chloroform-d) 𝛿 159.93, 155.26, 155.22, 138.35, 131.03, 130.00, 125.97, 120.91, 120.55, 119.75, 112.77, 108.53, 55.56, 47.08, 27.13. 7-Methoxy-9,9-dimethyl-9H-fluorene-2-carbaldehyde (8) To a solution of compound 7 (439 mg, 1.45 mmol) in dry THF, n-BuLi (1.81 mL of 1.6 M solution in hexane, 2.9 mmol) was added dropwise at -78 °C under nitrogen. The reaction mixture was stirred for 1 hour at the same temperature, and 281 an orange suspension was formed. DMF (212 mg, 2.9 mmol) was added dropwise, and the formed solution was stirred for additional 2 hour at -78 °C. Then the reaction mixture was warmed to room temperature and stirred for 1 hour. The reaction was quenched with 2 M HCl aq., and the solution was extracted three times with ethyl acetate. The combined organic phase was dried with Na2SO4 and evaporated. The crude was purified by flash chromatography (hexane/ ethyl acetate 96:4) to yield compound 8 as a white solid (230 mg, 63%). 1H NMR (500 MHz, Chloroform-d) 𝛿 10.03 (s, 1H), 7.94 (d, J = 1.4 Hz, 1H), 7.84 (dd, J = 7.8, 1.4 Hz, 1H), 7.75 (d, J = 7.8 Hz, 1H), 7.71 (d, J = 8.3 Hz, 1H), 7.00 (d, J = 2.3 Hz, 1H), 6.94 (dd, J = 8.4, 2.4 Hz, 1H), 3.91 (s, 3H), 1.50 (s, 6H). MR0 To a solution of compound 8 (50 mg, 0.2 mmol) in 3 mL dry DCM, BBr3 (0.5 mL of 1 M solution in DCM, 0.5 mmol) was added dropwise at 0 °C and let the reaction warm to room temperature under nitrogen. After 4 h, the reaction was quenched by ice water and extracted three times by DCM. The solvent evaporated and the product purified by flash chromatography (hexane/ ethyl acetate 88:12) to yield MR0 as a yellow solid (14.8 mg, 31%). 1H NMR (500 MHz, Chloroform-d) 𝛿 10.03 (s, 1H), 7.93 (d, J = 1.4 Hz, 1H), 7.84 (dd, J = 7.8, 1.5 Hz, 1H), 7.75 (d, J = 7.8 Hz, 1H), 7.67 (d, J = 8.2 Hz, 1H), 6.95 (d, J = 2.3 Hz, 1H), 6.87 (dd, J = 8.2, 2.4 Hz, 1H), 1.50 (s, 6H). 282 13C NMR (126 MHz, Chloroform-d) 𝛿 192.28, 157.52, 156.95, 153.76, 139.31, 134,53, 130.95, 130.65, 122.82, 122.48, 119.31, 114.79, 110.09, 46.87, 26.97. IV.7.5 Synthesis of MR1 (EtO)2OP CN 1.2 equiv. DIBAL, 3 equiv. O O O NaH, 1.8 equiv. CN THF, -78°C to r.t., THF, 0°C to r.t., overnight, 36% 8 4 h, 82% 9 BBr3, 2.5 equiv. O HO DCM, 0°C to r.t., O 4 h, 28% O 10 MR1 (E)-3-(7-methoxy-9,9-dimethyl-9H-fluoren-2-yl)acrylonitrile (9) A solution of diethyl (cyanomethyl)phosphonate (72 mg, 0.41 mmol) in 2 mL tetrahydrofuran was added to a stirred suspension of NaH (60% on mineral oil, 25 mg, 0.62 mmol) in 2 mL tetrahydrofuran at 0 °C. The mixture was stirred at this temperature for 30 min. Then a solution of compound 8 (85 mg, 0.34 mmol) in 3 mL tetrahydrofuran was added. The reaction was stirred at ambient temperature for 4 hours. The reaction mixture was then poured into cold water and extracted with ethyl acetate. The organic phase was dried over Na2SO4, then the solvent evaporated, and the product purified by flash chromatography (hexane/ ethyl acetate 94:6) to yield compound 9 (76mg, 82%). 1H NMR (500 MHz, Chloroform-d) 𝛿 7.65 (dd, J = 9.9, 8.1 Hz, 2H), 7.50 – 7.37 (m, 3H), 6.98 (d, J = 2.3 Hz, 1H), 6.92 (dd, J = 8.4, 2.3 Hz, 1H), 5.89 (d, J = 16.6 Hz, 1H), 3.90 (s, 3H), 1.49 (s, 6H). 283 13C NMR (126 MHz, Chloroform-d) 𝛿 160.56, 156.35, 153.96, 151.00, 142.68, 131.37, 130.82, 127.29, 121.62, 121.12, 119.62, 118.74, 113.10, 108.54, 94.36, 55.58, 46.90, 27.14. (E)-3-(7-methoxy-9,9-dimethyl-9H-fluoren-2-yl)acrylaldehyde (10) To a solution of compound 9 (44 mg, 0.16 mmol) in 3 mL dry tetrahydrofuran was added DIBAL (1 M in hexane, 0.5 mL) at -78 °C under argon. The reaction mixture was then allowed to warm to room temperature and was stirred overnight. Cold methanol was added dropwise to quench the reaction. The mixture was treated with saturate solution of Rochelle’s salt and extracted with dichloromethane. The organic phase was dried over Na2SO4, then the solvent evaporated, and the product purified by flash chromatography (hexane/ ethyl acetate 95:5) to yield compound 10 as yellow solid (16 mg, 36%). 1H NMR (500 MHz, Chloroform-d) 𝛿 9.72 (d, J = 7.7 Hz, 1H), 7.69 – 7.64 (m, 2H), 7.62 – 7.51 (m, 3H), 7.00 (d, J = 2.4 Hz, 1H), 6.93 (dd, J = 8.4, 2.4 Hz, 1H), 6.78 (dd, J = 15.8, 7.7 Hz, 1H), 3.90 (s, 3H), 1.51 (s, 6H). 13C NMR (126 MHz, Chloroform-d) 𝛿 193.78, 160.60, 156.48, 153.96, 153.58, 142.86, 131.87, 130.93, 128.56, 127.32, 122.32, 121.67, 119.66, 113.12, 108.55, 55.57, 46.89, 27.16. 284 MR1 To a solution of compound 10 (52 mg, 0.2 mmol) in 3 mL dry DCM, BBr3 (0.5 mL of 1 M solution in DCM, 0.5 mmol) was added dropwise at 0 °C and let the reaction warm to room temperature under nitrogen. After 4 h, reaction was quenched by ice water and extracted by DCM. The solvent evaporated and the product purified by flash chromatography (hexane/ ethyl acetate 9:1) to yield MR1 as a yellow solid (14.7 mg, 28%). 1H NMR (500 MHz, Chloroform-d) 𝛿 9.72 (d, J = 7.8 Hz, 1H), 7.68 – 7.52 (m, 5H), 6.95 (d, J = 2.3 Hz, 1H), 6.86 (dd, J = 8.2, 2.3 Hz, 1H), 6.79 (dd, J = 15.8, 7.8 Hz, 1H), 1.49 (s, 6H). 13C NMR (126 MHz, Chloroform-d) 𝛿 194.16, 156.83, 156.72, 154.05, 153.89, 142.95, 131.79, 130.91, 128.65, 127.18, 122.37, 121.86, 119.61, 114.68, 110.09, 46.83, 27.10. 285 IV.7.6 Synthesis of Me-TR1 CH3I, 2.4 equiv. KOH, 3.6 equiv. NBS, 2 equiv S S DMF, r.t., Br S S Br S S KI, 10 mol% 11 DMSO, 0°C to r.t., 12 2 h, 93% 13 16 h, 91% n-BuLi, 4 equiv. NaOMe, 10 equiv. DMF, 10 equiv. O O Br O CuO, 33 mol% S S THF/ Hexane, S S NaI, 5 mol% 14 -78°C to r.t., 15 THF, reflux, 15.5 h, 58% 48 h, 29% (EtO)2OP CN 1.2 equiv. DIBAL, 3.6 equiv. CN NaH, 3.2 equiv. O S S THF, -78°C to r.t., THF, 0°C to r.t., 16 3 h, 46% 4 h, 98% O O S S Me-TR1 4,4-dimethyl-4H-cyclopenta[2,1-b:3,4-b']dithiophene (12) Potassium hydroxide (1.12 g, 20 mmol) was added to a solution of 4H- cyclopenta[2,1-b:3,4-b']dithiophene 11 (1 g, 5.6 mmol), KI (93 mg, 0.56 mmol) and CH3I (1.9 g, 13.4 mmol) in DMSO (33 mL) at 0 °C. Then the reaction was stirred at room temperature for 16 hours. After completion the reaction, organic phase was extracted with diethyl ether and washed multiple times with water to decrease the amount of DMSO. Then, combined organic layers were dried over Na2SO4 and after evaporating the solvent, the product was purified via flash alumina (activated) column chromatography with hexane as eluent to give compound 12 (1.04g, 91%). 286 1H NMR (500 MHz, Chloroform-d) 𝛿 7.15 (d, J = 4.9 Hz, 1H), 6.99 (d, J = 4.9 Hz, 1H), 1.45 (s, 3H). 13C NMR (126 MHz, Chloroform-d) 𝛿 155.93, 137.24, 124.72, 122.17, 38.92, 25.24. 2,6-dibromo-4,4-dimethyl-4H-cyclopenta[2,1-b:3,4-b']dithiophene (13) A solution of 12 (1g, 4.85 mmol) and NBS (1.75 g, 9.83 mmol) in 35 mL dry DMF (distilled and dried on molecular sieve) was stirred in dark at room temperature for 2 hours. The reaction mixture was poured into 50 mL of saturated sodium thiosulfate aqueous solution, and the product was extracted three times with hexane. The combined organic layers were washed with saturated sodium chloride solution and dried over Na2SO4 and filtered. The filtrate was concentrated and purified via flash alumina (activated) column chromatography by using hexane as eluent to give compound 13 as white solid (1.64 g, 93%). 1H NMR (500 MHz, Chloroform-d) 𝛿 7.00 (s, 2H), 1.41 (s, 6H). 13C NMR (126 MHz, Chloroform-d) 𝛿 158.30, 135.13, 123.96, 111.37, 46.42, 24.82. 2-bromo-6-methoxy-4,4-dimethyl-4H-cyclopenta[2,1-b:3,4-b']dithiophene (14) Sodium methoxide was prepared by adding sodium (150 mg, 6.5 mmol) into 3 mL of anhydrous methanol under nitrogen. When the sodium disappeared, a solution of compound 13 (236 mg, 0.65 mmol) in 2 mL of dry THF, copper oxide (17.8 mg, 0.22 mmol) and a catalytic amount of sodium iodide was added to the above solution and heated to reflux for 48 hours. The reaction mixture was poured 287 into water and then extracted with DCM. The organic layer was washed with brine and dried over Na2SO4. The crude was purified by alumina (activated) column chromatography (hexane/ ethyl acetate 99:1) to give compound 14 (59mg, 29%). 1H NMR (500 MHz, Chloroform-d) 𝛿 6.98 (s, 1H), 6.23 (s, 1H), 3.93 (s, 3H), 1.39 (s, 6H). 13C NMR (126 MHz, Chloroform-d) 𝛿 167.77, 156.08, 155.65, 136.31, 123.79, 120.06, 108.55, 99.36, 60.84, 46.76, 24.91. 6-methoxy-4,4-dimethyl-4H-cyclopenta[2,1-b:3,4-b']dithiophene-2- carbaldehyde (15) To a solution of compound 14 (438 mg, 1.39 mmol) in dry THF, n-BuLi (2.2 mL of 2.5 M solution in hexane, 5.56 mmol) was added dropwise at -78 °C under nitrogen. The reaction mixture was stirred for 1.5 hour at the same temperature, and an orange suspension was formed. DMF (1 mL) was added dropwise, and the formed solution was stirred for an additional 2 hour at -78 °C. Then the reaction mixture was warmed to room temperature and stirred for 12 hours. The reaction was quenched with water and was extracted three times with DCM. The combined organic phase was dried with Na2SO4 and evaporated. The crude was purified by flash alumina (activated) column chromatography (hexane/ ethyl acetate 8:2) to yield pure compound 15 (212 mg, 58%). 1H NMR (500 MHz, Chloroform-d) 𝛿 9.77 (s, 1H), 7.57 (s, 1H), 6.28 (s, 1H), 3.98 (s, 3H), 1.46 (s, 6H). 288 (E)-3-(6-methoxy-4,4-dimethyl-4H-cyclopenta[2,1-b:3,4-b']dithiophen-2- yl)acrylonitrile (16) A solution of diethyl (cyanomethyl)phosphonate (155 mg, 0.87 mmol) in 4 mL tetrahydrofuran was added to a stirred suspension of NaH (60% on mineral oil, 94 mg, 2.35 mmol) in 4 mL tetrahydrofuran at 0 °C. The mixture was stirred at this temperature for 30 min. Then a solution of compound 15 (193 mg, 0.73 mmol) in 4 mL tetrahydrofuran was added. The reaction mixture was stirred at ambient temperature for 4 hours. The reaction mixture was then poured into cold water and extracted with ethyl acetate. The organic phase was dried over Na2SO4, then the solvent evaporated, and the product purified by flash alumina (activated) column chromatography (hexane/ ethyl acetate 85:15) to yield compound 16 as a yellow solid (205 mg, 98%). 1H NMR (500 MHz, Chloroform-d) 𝛿 7.40 (dd, J = 16.0, 0.6 Hz, 1H), 7.07 (s, 1H), 6.25 (s, 1H), 5.44 (d, J = 16.0 Hz, 1H), 3.96 (s, 3H), 1.41 (s, 6H). 13C NMR (126 MHz, Chloroform-d) δ 170.34, 159.91, 157.99, 143.47, 141.78, 136.49, 124.78, 119.70, 119.27, 99.34, 89.06, 60.69, 46.24, 24.86. Me-TR1 To a solution of compound 16 (145 mg, 0.50 mmol) in 9 mL dry tetrahydrofuran was added DIBAL (1 M in hexane, 1.8 mL) at -78 °C under argon. The reaction mixture was then allowed to warm to room temperature and was stirred for 3 hours. Cold methanol was added dropwise to quench the reaction. The mixture was treated with saturate solution of Rochelle’s salt and extracted with 289 dichloromethane. The organic phase was dried over Na2SO4, then the solvent evaporated, and the product purified by flash alumina (activated) column chromatography (hexane/ ethyl acetate 8:2) to yield Me-TR1 as yellow solid (66 mg, 46%). 1H NMR (500 MHz, Chloroform-d) 𝛿 9.56 (d, J = 7.8 Hz, 1H), 7.53 (dd, J = 15.4, 0.6 Hz, 1H), 7.20 (s, 1H), 6.41 (d, J = 15.4 Hz, 1H), 6.26 (s, 1H), 3.97 (s, 3H), 1.44 (s, 6H). 13C NMR (126 MHz, Chloroform-d) 𝛿 192.57, 170.64, 160.19, 158.41, 145.85, 143.76, 137.48, 125.72, 123.40, 119.93, 99.36, 60.68, 46.20, 24.92. IV.7.7 Synthesis of CyThioPhenol methyl 5-methoxy-2-(thiophen-2-yl)benzoate (18) To the mixture of methyl 2-iodo-5-methoxybenzoate 17 (292 mg, 1.0 mmol), Pd(PPh3)4 (116 mg, 0.1 mmol), and toluene (20.0 mL), a solution of thiophen-2- ylboronic acid (384 mg, 3 mmol) in ethanol (5 mL) and K2CO3 (1.38 g, 10 mmol) in water (2.5 mL) were added. The mixture was heated to 80 °C in an argon atmosphere for 2 hours. After cooling to room temperature, the mixture was filtered through Celite, and the organic phase was washed with water. The organic layers were then dried over Na2SO4, filtered, and concentrated under vacuum. Column chromatography with (hexane/ ethyl acetate 19:1) of the residue afforded compound 18 as a colorless oil (143 mg, 58%). 290 1H NMR (500 MHz, Chloroform-d) 𝛿 7.41 (d, J = 8.5 Hz, 1H), 7.31 (dd, J = 5.1, 1.2 Hz, 1H), 7.26 (d, J = 2.8 Hz, 1H), 7.07 – 7.01 (m, 2H), 6.97 (dd, J = 3.5, 1.2 Hz, 1H), 3.87 (s, 3H), 3.74 (s, 3H). 13C NMR (126 MHz, Chloroform-d) 𝛿 168.86, 159.04, 141.99, 132.53, 127.09, 126.58, 125.86, 125.33, 117.17, 114.21, 55.57, 52.29. CO2CH3 S B(OH)2 CO2CH3 1) CH3MgBr, 3 equiv. 3 equiv. THF, overnight, 1.5 h O I O Pd(PPh3)4, 10 mol % S 2) AcOH, H2SO4 (4:1) 17 K2CO3, 10 equiv. 18 6 h, 120°C, toluene/ EtOH/ H2O, two steps 14% (20:5:2.5), 80°C, 2 h, 58% O O EtO P EtO OEt O O 24 equiv. O O S NaH, 24 equiv. S OEt 19 THF, 0°C to reflux, 20 36 h, 43% DIBALH, 3 equiv. DMP, 1.4 equiv. O OH DCM, -78°C, 1 h NaHCO3, 1.6 equiv. S DCM, 0°C, 1 h 21 two steps 53% O O BBr3, 2 equiv. HO O S DCM, 0°C to r.t., S 22 3 h, 37% CyThioPhenol 1-(6-methoxy-4,4-dimethyl-4H-indeno[1,2-b]thiophen-2-yl)ethan-1-one (19) 291 In a flame dried and under argon atmosphere, compound 18 (248 mg, 1.0 mmol) was dissolved in 5 mL dry THF and methyl magnesium bromide (1M in THF, 3.5 mL, 3.5 mmol) were added dropwise over 1.5 hours at room temperature. The reaction mixture was stirred under argon overnight at same temperature. The reaction was quenched with 1M HCl and extracted by DCM. The organic layer was then dried over Na2SO4 and concentrated under vacuum. The tertiary alcohol product was used for the next step without further purification. A mixture of 4:1 glacial acetic acid and 98% sulfuric acid (7 mL) was added, and the mixture heated at 120 °C for 6 hour. After cooling the reaction, it was quenched with 25 M solution of ammonia and extracted with DCM three times. The organic layers were then dried over Na2SO4, filtered, and concentrated under vacuum. Column chromatography with (hexane/ DCM 5:1) of the residue afforded pure compound 19 (38 mg, 14%). 1H NMR (500 MHz, Chloroform-d) 𝛿 7.61 (d, J = 0.7 Hz, 1H), 7.46 (d, J = 8.3 Hz, 1H), 6.95 (d, J = 2.4 Hz, 1H), 6.86 (dd, J = 8.3, 2.3 Hz, 1H), 3.88 (s, 3H), 2.57 (d, J = 0.7 Hz, 3H), 1.48 (s, 6H). 13C NMR (126 MHz, Chloroform-d) 𝛿 190.70, 159.82, 159.16, 157.11, 149.03, 144.92, 128.34, 126.28, 121.40, 112.45, 109.29, 55.62, 45.91, 26.58, 26.23. ethyl (E)-3-(6-methoxy-4,4-dimethyl-4H-indeno[1,2-b]thiophen-2-yl)but-2- enoate (20) 292 Triethyl phosphonoacetate (1.98 mL, 10 mmol) was added dropwise under nitrogen to a suspension of NaH (60% on mineral oil, 400 mg, 10 mmol) in dry THF (5mL) at 0 °C and the resulting mixture was stirred for another 30 min at the same temperature. A solution of compound 19 (170 mg, 0.62 mmol) in 2 mL THF was added dropwise to the reaction mixture and heated to reflux. The reaction followed by TLC and after 24 hours, the reaction was not complete. Then the reaction mixture was cooled to 0 °C and NaH (60% on mineral oil, 200 mg, 5 mmol) and triethyl phosphonoacetate (0.99 mL, 5 mmol) were added to the mixture and heated to reflux for 12 hours. After completion of the reaction, the mixture was brought to room temperature and quenched with saturated NH4Cl and extracted three times with ethyl acetate. The organic layers were then dried over Na2SO4 and concentrated under vacuum. Column chromatography with (hexane/ ethyl acetate 96:4) of the residue afforded a mixture of trans/cis (2:1) isomers of compound 20 (91 mg, 43%). (E)-3-(6-methoxy-4,4-dimethyl-4H-indeno[1,2-b]thiophen-2-yl)but-2-en-1-ol (21) To the mixture of cis/trans isomers of compound 20 (127 mg, 0.37 mmol) in dry DCM (10 mL) at -78 °C was added DIBALH (1M in hexane, 1.1 mL, 1.1 mmol) dropwise. After 1 hour, the reaction was complete, and methanol (5 mL) was added dropwise to quench the reaction. Then 15 mL of saturated solution of potassium sodium tartrate was added with 10 mL of DCM. The mixture was stirred for 2 h and extracted with DCM (3 × 10 mL) and the combined organic layers were dried over 293 Na2SO4. Evaporation of solvent gave compound 21 as a yellow solid and it used in next step without further purification. (E)-3-(6-methoxy-4,4-dimethyl-4H-indeno[1,2-b]thiophen-2-yl)but-2-enal (22) To compound 21 (used directly from last step) in dry DCM (10 mL) at 0 °C, Dess-Martin periodinane (DMP) (207 mg, 0.5 mmol) and NaHCO3 (50 mg, 0.6 mmol) were added. The reaction mixture was stirred at this temperature for 1 hour and after completion of reaction it was quenched by saturated sodium thiosulfate (10 mL) and saturated sodium bicarbonate (10 mL). The mixture was extracted with DCM (3 × 10 mL) and the combined organic layers were dried over Na2SO4. After evaporating the solvent, the product was purified via column chromatography by using (hexane/ ethyl acetate 9:1) as eluent to give compound 22 as a mixture of trans/cis (2:1) isomers (58 mg, two steps 53%). CyThioPhenol To a solution of compound 22 (89 mg, 0.3 mmol, mixture of trans/cis 2:1) in 3 mL dry DCM, BBr3 (0.6 mL of 1 M solution in DCM, 0.6 mmol) was added dropwise at 0 °C and let it warm to room temperature under nitrogen. After 3 h, reaction was quenched by ice water and extracted by DCM. The solvent was evaporated, and it purified by flash chromatography (hexane/ ethyl acetate 9:1) to yield trans CyThioPhenol as an orange solid (31.5 mg, 37%). 294 1H NMR (500 MHz, Acetone-d6) 𝛿 10.13 (d, J = 7.7 Hz, 1H), 8.63 (s, 1H), 7.72 (s, 1H), 7.39 (d, J = 8.1 Hz, 1H), 7.01 (d, J = 2.2 Hz, 1H), 6.83 (dd, J = 8.2, 2.3 Hz, 1H), 6.35 (dq, J = 7.7, 1.1 Hz, 1H), 2.64 (d, J = 1.1 Hz, 3H), 1.47 (s, 6H). 13C NMR (126 MHz, Acetone-d6) 𝛿 189.67, 158.97, 157.73, 157.42, 150.50, 144.84, 143.71, 127.31, 122.93, 122.03, 120.66, 114.17, 110.49, 45.75, 25.37, 15.06. 295 REFERENCES 296 REFERENCES 1 Wang, W. In PhD Thesis. Michigan State University (2012). 2 Wang, W. et al. Tuning the electronic absorption of protein-embedded all- trans-retinal. Science 338, 1340-1343, doi:10.1126/science.1226135 (2012). 3 Gill, S. C. & von Hippel, P. H. Calculation of protein extinction coefficients from amino acid sequence data. Analytical biochemistry 182, 319-326 (1989). 4 Yapici, I. et al. "Turn-On" Protein Fluorescence: In Situ Formation of Cyanine Dyes. Journal of the American Chemical Society 137, 1073-1080, doi:10.1021/ja506376j (2015). 5 Yapici, I. In PhD Thesis. Michigan State University (2015). 6 Santos, E. M. In PhD Thesis. Michigan State University (2017). 297