4...... to. . z .5: :l. (1-: t g 13.3. . .. It! a 5. . a . 1. Nu 3. Eu. . J . t . :. If“?! .31 1.. 1.1.. I; ‘39. ‘ 5.5. x . . 5 1.. A: t. r. uh! ‘ $13!. £31.51». ‘ fl 4:. H39 ~.. .eh... 21.... I —-§I 13" r11... aw... I. t. I». 1 .‘rflfv‘d? i: . , 2.... I. , . .2 .2 if... . $511.5: 4. 1.0.3.922; [in 3.3.6 I..- h I. .30.. wiplnmn. . ffi 31-: . 5 . 2., s 13.: ” '4- r‘l» . 5.5,: iii... . 0.: , II It, 1.. I. 1.9;.” :‘3. , I! is z r... ... :t J J { #3,: ’ $5,; : sagtjéfi, This is to certify that the dissertation entitled SPORE PHOTOPRODUCT LYASE: INVESTIGATIONS OF A RADICAL DNA REPAIR ENZYME presented by Jeffrey Michael Buis has been accepted towards fulfillment of the requirements for the PhD. degree in Chemistry a? 7 Major Professor’s Signature Date MSU is an Affirmative Action/Equal Opportunity Institution LER—ARY Michigan State University PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 2/05 p:/ClRC/DateDue.indd-p.1 SPORE PHOTOPRODUCT LYASE: INVESTIGATIONS OF A RADICAL DNA REPAIR ENZYME By Jeffrey Michael Buis A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Chemistry 2006 ABSTRACT SPORE PHOTOPRODUCT LYASE: INVESTIGATIONS OF A RADICAL DNA REPAIR ENZYME By Jeffrey Michael Buis Exposure to ultra—violet light in sporulating bacteria creates a unique form of DNA damage known as the spore photoproduct (SP). Sporulating bacteria contain a novel direct reversal repair enzyme to repair this damage called spore photoproduct lyase (SPL), which catalyzes the repair of SP dimers to thymine monomers. Presented here is the first detailed characterization of catalytically active SPL, which has been anaerobically purified from overexpressing Escherichia coli. Anaerobically purified SPL is monomeric and is red-brown in color. The purified enzyme contains ~3.1 iron and 3.0 acid labile 82' per protein and has a UV-visible spectrum characteristic of an iron sulfur proteins ((410 nm 11.9 mM'1 cm’1) and 450 nm (10.5 mM'1 cm")). The X-band EPR spectrum of the purified enzyme shows a nearly isotropic signal (9 = 2.02) characteristic of a [3Fe-4S]1+ cluster; reduction of SPL with sodium dithionite results in the appearance of a new EPR signal (9 = 2.03, 1.93, and 1.89) consistent with assignment to a [4Fe-4S]1+ cluster. Mossbauer results confirm the presence of a mixture of cluster states in the isolated protein with assignment to [2Fe-28]2+ and [3Fe—4S]1+ clusters. Reduction with sodium dithionite yields primarily a [4Fe- 48]“ cluster. The reduced purified enzyme is active in SP repair, with a specific activity of 0.33 umol/min/mg. This can be increased to 1.33 umol/min/mg by using SPL that has been reduced prior to the assay and correlates to 2.4 umol/min/mg of SPL with a [4Fe-4S]1+ cluster. Only a catalytic amount of SPL is required for DNA repair and no irreversible cleavage of S-adenosylmethionine into methionine and 5’-deoxyadenosine is observed during the reaction. Label transfer from [5’-3H] S-adenosylmethionine to repaired thymine is observed, providing evidence to support a mechanism in which a 5’-deoxyadenosyl radical intermediate directly abstracts a hydrogen from SP 0-6 to generate a substrate radical, and subsequent to radical mediated B-scission, a product radical abstracts a hydrogen from 5’-deoxyadenosine to regenerate the 5’- deoxyadenosyl radical. An isotope affect of ~15-17 can be calculated from 0-6 - 3H label transfer experiments, suggesting initial H atom abstraction to be a slow step in the repair reaction. Gel shift assays show SPL binding DNA in the presence and absence of the iron sulfur cluster. This work is dedicated to my beautiful wife Lorraine Buis who has loved and supported me during all of the endeavors of my life and has put up with me no matter what. I never would have made it without you and look fonivard to many more adventures together. I would also like to dedicate this work to my parents John and Judith Buis who have always been there to help me in anyway possible. iv ACKNOWLEDGEMENTS I would like to express my thanks to my advisor Dr. Joan Broderick, who has guided me throughout my graduate career. Without her advice and support, I never would have achieved this goal. I would also like to acknowledge my graduate committee members, Dr. James McCusker, Dr. Babak Borhan and Dr. David Weliky. I would like to thank Dr. Danilo Ortillo, whose help with EPR spectroscopy has proved indispensable. I would also like to thanks him for his years of friendship and a place to stay while I finished my research. I would like to acknowledge Dr. Jennfier Cheek for her training me in the field of biochemistry and teaching me the intricacies of the SPL project. I thank Dr. Mbako Nyepi whom I miss very much and hope to visit someday in Botswana. Furthermore I would like to acknowledge Efthalia Kalliri for providing me with the small,acid soluble protein necessary for these experiments. I would like to acknowledge Dr. Will Broderick for help in setting up the lab again among other things. I would like to give my thanks to my collaborators on the Mossbauer portion of this project, Dr. Vincent Huyhn and Dr. Ricardo Garcia. I would also like to thank Dr. Brian Hoffman and Dr. Nick Lees for their work on the ENDOR experiments. I would like to thanks all of my former lab mates, Egidijus Zilinskas, Yi Peng, Jian Yang, Magdalena Makowska-Grzyska, Ziyang Su, Meng Li, Shujaun Xu, Kaitlin Duschene, Sunshine Silver, Rachel Udelhoven, Tilak Chandra, Chris Austin, Brian Facione and everyone else whose presence had made this such great experience for me. vi TABLE OF CONTENTS LIST OF TABLES ................................................................................................ xiii LIST OF SCHEMES.............................. .............................................................. xv LIST OF FIGURES ............................................................................................. xvi LIST OF ABBREVIATIONS ................................................................................ xx CHAPTER I INTRODUCTION .................................................................................................. 1 L1 Iron-Sulfur Clusters in Biology .......................................................................... 1 l.2 DNA Damage, Spore Photoproduct and Small Acid Soluble Protein ............... 4 L3 Spore Photoproduct Lyase .............................................................................. 7 L4 Radical SAM Superfamily ............................................................................. 10 L5 Recent Work on Spore Photoproduct Lyase ................................................. 18 LG References................. .................................................................................... 23 CHAPTER II OVEREXPRESSION, PURIFICATION OF SPORE PHOTOPRODUCT LYASE32 ”.1 Introduction ................................................................................................... 32 ".2 Experimental Methods .................................................................................. 34 Materials ............................................................................................................. 34 Growth of spore photoproduct lyase .................................................................. 34 Purification of spore photoproduct lyase ............................................................ 35 Protein, iron and sulfide assays ......................................................................... 36 Gel filtration chromatography ............................................................................. 36 ".3 Results and Discussion ................................................................................ .37 Purification of spore photoproduct lyase ............................................................. 37 vii Subunit structure of spore photoproduct lyase .................................................... 39 ".4 Conclusions .................................................................................................. 42 ".5 Refences ....................................................................................................... 43 CHAPTER III SOLUBILIZATION OF SPORE PHOTOPRODUCT LYASE .............................. 45 ”L1 Introduction .................................................................................................. 45 III.2 Experimental Methods ................................................................................. 47 Materials ............................................................................................................ 47 Lysis procedures ................................................................................................ 47 Cloning Bacillus subtilus spIB into pET28a, pET 42a and pET44a .................... 49 Cloning spIB from Bacillus halodurans into pET30/EK/LIC ................................ 50 Transformation of pET14B into various competent cell strains .......................... 50 Protein dialysis and concentration for varying buffer conditions ......................... 50 Altered growth conditions of pET14b—spl17in Tuner(DE3)pLysS E. coli ............ 51 Protein and iron assays ..................................................................................... 54 "L3 Results and Discussion ................................................................................ 54 Cloning Bacillus subtilus spIB into pET28a, pET 42a and pET44a .................... 54 Cloning splB from Bacillus halodurans into pET30/EK/LIC ................................ 57 Overexpression of splB in Rosetta and CodonPlus competent cells .................. 61 Differing buffer conditions of the pET14b/spl17 construct .................................. 62 Effect of altered growth conditions on overexpression of pET14b-spl17 ............ 63 "L4 Conclusions ................................................................................................. 64 llI.5 References .................................................................................................. .66 viii CHAPTER IV SPECTROSCOPIC CHARACTERIZATION OF THE IRON SULFUR CLUSTER IN SPORE PHOTOPRODUCT LYASE ............................. 67 N1 Introduction ..................................... i ............................................................. 67 lV.2 Experimental Methods ................................................................................ .72 Materials ............................................................................................................ 72 Synthesis of S-adenosyl-L-methionine ............................................................... 72 Growth and Purification of 57Fe spore photoproduct lyase ................................ 73 UV/visible spectroscopy ..................................................................................... 73 Electron paramagnetic spectroscopy ................................................................ .74 Mossbauer spectroscopy ................................................................................... 74 IV.3 Results and Discussion ............................................................................... 75 Synthesis of S-adenosyl-L-methionine ............................................................... 75 UV/visible spectroscopy ..................................................................................... 76 Electron paramagnetic spectroscopy ................................................................. 77 Mossbauer spectroscopy ................................................................................... 81 N4 Conclusions ................................................................................................. 83 NS References .................................................................................................. 86 CHAPTER V ENZYMATIC ACTIVITY OF SPORE PHOTOPRODUCT LYASE ...................... 88 V.1 Introduction ................................................................................................... 88 V2 Experimental Methods .................................................................................. 90 Materials ............................................................................................................ 90 3H labeling and synthesis of the spore photoproduct.....................................90 Time course repair assays of spore photoproduct ............................................. 91 ix Repair assay with pre-reduced SPL ................................................................... 92 V.3 Results and Discussion ................................................................................ 93 V.4 Conclusions .................................................................................................. 98 V5 References ................................................................................................. 100 CHAPTER VI MECHANISTIC CONSIDERATIONS FOR SPORE PHOTOPRODUCT LYASE102 Vl.1 Introduction ................................................................................................ 102 Vl.2 Experimental Methods ............................................................................... 106 Materials .......................................................................................................... 1 06 Synthesis of [2, 5', 8 -3H] SAM ......................................................................... 106 Production of unlabeled spore photoproduct ................................................... 107 Production of [C-6 - 3H] thymine labeled spore photoproduct .......................... 108 Tritium transfer from SAM to thymine .............................................................. 108 Tritium transfer from thymine to SAM .............................................................. 109 SAM cleavage by spore photoproduct lyase .................................................... 110 Vl.3 Results and Discussion ............................................................................. 111 SAM is not cleaved to 5’-deoxyadenosine during SP repair ............................ 111 Direct H atom transfer from [2, 5’, 8 -3H] SAM to repaired thymine ................. 114 A potential isotope effect for H atom abstraction .............................................. 116 Vl.4 Conclusions ............................................................................................... 120 Vl.5 References ................................................................................................ 122 CHAPTER VII DNA BINDING PROPERTIES OF SPORE PHOTOPRODUCT LYASE .............................................................................. 124 VI|.1 Introduction ............................................................................................... 124 Vll.2 Experimental Methods .............................................................................. 125 32F End labeling of 94mer oligonucleotide ....................................................... 125 Gel shift DNA binding assay ............................................................................ 127 Preparation of apo-SPL ................................................................................... 131 VII.3 Results and Discussion ............................................................................ 131 DNA binding of as isolated protein ................................................................... 131 DNA binding of SPL under aerobic conditions ................................................. 133 DNA binding of apo-SPL .................................................................................. 135 DNA binding of as isolated protein with SAM ................................................... 138 V". Conclusions ............................................................................................... 139 Vll.5 Refences .................................................................................................. 142 CHAPTER VIII INTERACTION OF S’ADENOSYLMETHIONINE AND THE IRON SULFUR CLUSTER OF SPORE PHOTOPRODUCT LYASE ......................................... 144 VII|.1 Introduction .............................................................................................. 144 VlI|.2 Experimental Methods ............................................................................. 146 Materials .......................................................................................................... 146 Synthesis of [2, 8, - 3H] SAM ................................................................................. Equilibrium dialysis of spore photoproduct lyase ............................................. 147 Electron nuclear double resonance spectroscopy and Q-band EPR sample Preparation ........................................................................................... 147 xi Q-band electron paramagnetic resonance ....................................................... 148 Electron nuclear double resonance spectroscopy ............................................ 148 Vlll.3 Results and Discussion ........................................................................... 150 Determination of the SAM-SPL dissociation constant ...................................... 150 Initial results from Q-band EPR of reduced SPL .............................................. 151 Initial ENDOR results ....................................................................................... 151 VIII.4 Conclusions ............................................................................................. 154 VIII.5 References .............................................................................................. 155 CHAPTER IX GENERAL CONCLUSIONS AND FUTURE DIRECTIONS .............................. 157 lX.1 The Iron Sulfur Cluster of SPL ................................................................... 157 lX.2 Catalytic Activity of SPL ............................................................................. 159 IX.3 Mechanism of SPL .................................................................................... 160 IX.4 Interaction of DNA and SPL ....................................................................... 162 IX.5 An Overview of Spore Photoproduct Repair .............................................. 163 IX.6 Implications for DNA Repair and Similarities to DNA Photolyase .............. 164 |X.7 Future Experiments: Protein Crystallography ............................................ 165 IX.8 Future Experiments: Synthesis of Synthetic Spore Photoproduct ............. 166 lX.9 Future Experiments: EXAFS studies of SPL .............................................. 167 IX.10 Future Experiments: DNA Binding Studies .............................................. 168 IX.11 References .............................................................................................. 169 xii LIST OF TABLES Table |.1. C-terminal sequence homology between DNA photolyases from different organisms (above) and spore photoproduct lyase from Bacillus subtilis (below) shows several conserved residues. ......................................................... 8 Table l.2. CX3CX2C conserved binding motif of the radical SAM superfamily....10 Table III.1. Overexpression and solubility results from the various expression vectors, competent cells and organisms. ............................................................ 58 Table III.2. The variance of buffering conditions and its affect on SPL. Buffer. 50 mM Hepes, 300 mM NaCI, 5 mM B-ME, 10% glycerol, pH 7.5, ~125 mM imidizole, gives a best concentration of 150 pM before precipitation. The increase in glycerol concentration was one of the few effective ways in raising the solubility of SPL. However, addition of SAM was most effective at stabilizing SPL. .................................................................................................................... 62 Table III.3. Amount of SPL present in whole cell lysis on SDS-PAGE gels as a percentage of the total cellular protein. ............................................................... 63 Table IV.1. Typical spectroscopic properties of iron sulfur clusters. ................... 68 Table IV.2. Percentage of total iron present in the iron sulfur cluster of SPL as monitored by EPR and Mdssbauer spectroscopy. .............................................. 84 Table Vl.1. Summary of SAM cleavage during SP repair reaction ................... 113 Table Vl.2. Summary of label transfer from 0-6 to SAM at increasing SPL repair reaction times and the corresponding isotope effect. ........................................ 119 Table VII.1. Reaction conditions for the gel shift assay of SPL and DNA under anaerobic conditions ......................................................................................... 127 Table Vll.2. Reaction conditions for the gel shift assay of SPL and DNA under aerobic conditions. ............................................................................................ 128 Table VII.3. Reaction conditions for the gel shift assay of SPL and DNA under anaerobic conditions with Apo-SPL. ................................................................. 129 Table Vll.4. Reaction conditions for the gel shift assay of SPL and DNA under anaerobic conditions with AdoMet .................................................................... 130 Table VI|.5. Percentage of bound protein as calculated from the band density of the gel shift assay. ............................................................................................ 140 xiii Table Vlll.1. Table of dissociation constants calculated from varying amounts of SPL during equilibrium dialysis. This yielded an average Kd = 200 pM i: 25 pM.150 xiv LIST OF FIGURES Figure L1. The three major categories of iron sulfur clusters are illustrated including the [4Fe-4S], [3Fe—4S], [2Fe-28] from left to right. ................................. 2 Figure ".1. SDS-PAGE of the purification of SPL from the metal affinity column chromatography, standards are shown on the left in lane 1. Lane 2 shows uninduced cells, lane 2 shows cell after addition of 1 mM IPTG to induce the cell culture. Lane 4 shows protein after purification on a Co-sepharose column. ...... 38 Figure ".2. FPLC chromatogram of SPL purification using cobalt metal affinity chromatography. SPL elutes ~20 min at 50% during an imidazole step gradient as a brownish-red band with a high absorbance at both 280 nm and 426 nm. ...39 Figure ".3. SDS-PAGE gel electrophoresis of crude extract and fractions eluting from the Co-sepharose column. Lane 1: Molecular weight standard. Lane 2: Crude Iysate before loading onto iMAC column. Lane 3-5: 1 mL fractions from Co-sepharose columns. ...................................................................................... 40 Figure ".4. Gel filtration chromatography of a SPL on a sepharoseTM 12 column. SPL loaded on a gel filtration column and run with isocratic flow shows partially purified SPL elutes primarily as a single band with a molecular weight of 46kDa. A slight shoulder at ~ 80kDa can be assigned to a small amount of dimeric protein. ................................................................................................................ 41 Figure Ill.1. SDS-PAGE of SPL from B. subtilus cloned into pET44a, pET42a, pET28a. A. The overexpression of pET44a-spl1, lanes 1 and 2 are uninduced cell cultures. Lanes 3 and 4 show induced cell cultures (whole cell lysis, procedure 1) to contain a large band at ~66 kDa. Lanes 5 and 6 are induced cell cultures enzymatically Iysed (lysis procedure 4). Lane 7 is the BioRad broad range protein standard. Lanes 8 and 9 are cells enzymatically Iysed (lysis procedure 2) B. The growth and whole cell lysis of pET28a-spl1 and pET42a-spl1 shows no significant overexpression of any band at the expected molecular weights of ~43 kDa and 55 kDa respectively; Lanes 1 is uninduced pET28a-spl1 cells and lane 2 is induced cells (growth condition 3, lysis procedure 1). Lanes 1 is uninduced pET42a-spl1 cells and lane 2 is induced cells (growth condition 3, lysis procedure 1) ................................................................................................ 56 Figure III.2. SDS-PAGE of SPL from B. halodurans overexpressed and Iysed. A. The addition of IPTG, lanes 1, 3, and 5, causes significant overexpression versus cultures (lanes 2, 4, 6) without IPTG (lysis procedure 1). Lane 7 is the BioRad protein standard. B. After cell lysis (procedure 4) no overexpressed protein is found in the supernatant, lanes 3 and 4. Most of the protein is found in the cell pellet, lanes 1 and 2. Lane 5 is the BioRad protein standard. ............................. 60 xvi Figure III.3. SDS-PAGE gel of pET14b-spl17 transformed and overexpressed in Rosetta and CodonPlus competent cells. Lane 1 and 2, induced and uninduced SPL in Rosetta competent cells. Lane 3 and 4, induced and uninduced SPL in Codon Plus competent cells. Lane 5, BioRad broad range protein Standard. ....61 Figure IV.1. Purification of SAM by cation exchange chromatography. SAM elutes as a large broad peak (A260) between approximately 0.4 and 0.6 M HCI on a source 155 cationic exchange column with a linear gradient between MQ H20 and 1M HCI. ........................................................................................................ 76 Figure IV.2. UV-visible absorption spectra of SPL as isolated (solid line) and reduced with dithionite (dashed line). For both spectra, the protein was 65 pM in 20 mM sodium phosphate/500 mM NaCI/5 mM dithiothreitoI/5% glycerol, pH 8.0. The reduced protein also contained 5 mM dithionite. The spectra were recorded in a 1 cm pathlength cuvette under anaerobic conditions at room temperature..77 Figure IV.3. X-band EPR spectrum of anaerobically isolated SPL. The protein was 350 pM in 20 mM sodium phosphate/500 mM NaCl/10 mM dithiothreitoI/5% glycerol, pH 8.0. Conditions of measurement, T=12 K microwave power, 2 mW; microwave frequency, 9.4841 GHz; modulation amplitude, 10.084; and receiver gain, 2 x 104, 1 scan accumulated. ..................................................................... 78 Figure IV.4. X-band EPR spectrum of reduced SP lyase with and without AdoMet. The protein was 350 uM in 20 mM sodium phosphate/500 mM NaCl/10 mM dithiothreitol/5% glycerol, pH 8.0. Dithionite was added to 5 mM (both spectra) and AdoMet to 2 mM (lower spectrum only). Conditions of measurement, T = 12 K microwave power, 2 mW; microwave frequency, 9.4841 GHz; modulation amplitude, 10.084; and receiver gain, 2 x 104, 1 scan accumulated ........................................................................................................ 79 Figure IV.5 Mbssbauer spectra of SPL. A. Native state SPL. The dashed black line is the actual experimental spectrum. The light blue line is a simulated spectrum of a [4Fe-4S]2+ cluster, weighted to 53% of the iron and the black line is the subtraction of the dashed black line and the light blue line. The remaining is indicative of a [3Fe-4S]1+ cluster. B. Reduced SPL. The dashed black line the experimental spectrum, overlaid on this spectra (black line) is the addition of two simulated clusters, the blue line being a [4Fe-4S]2+ cluster and the pink being a [4Fe-4S]1+ cluster weighted to 10% and 90% of the total iron. C. Reduced SPL with SAM (dashed black line) and a simulation for a [4Fe-S]2+ cluster (Blue line) weighted to 33% of the iron. The black line is the subtraction of the two above spectra, and is indicative of a [4Fe-4S]1+ cluster. ................................................ 82 Figure V.1. The separation of thymine and SP after HPLC is illustrated above. The solid line is UV-irradiated and hydrolyzed DNA without the addition of SPL. The dashed line is UV-irradiated and hydrolyzed DNA after the addition of SPL xvii and incubated for 60 min. Both samples were acid hydrolyzed and loaded onto a Waters Spherisorb SSP column and run with an isocratic flow of degassed MQ water for 25 minutes at 1.8 mL/min. Fractions were collected every minute and run on a liquid scintillation counter. ..................................................................... 93 Figure v.2. A representation of SP repair over a time of 60 min. Thymine elutes at ~ 3 min, and SP elutes at ~10 min. Over the course of 60 minutes the amount of SP decreases 70%. ........................................................................................ 94 Figure v.3. SP lyase is active in SP repair. Representative time course of SP repair by reduced SP lyase. Repair of pUC18 DNA was monitored at 10 min intervals by removal of 100 pL aliquots, which were quenched hydrolyzed, and monitored by HPLC for SP repair. Linear repair is observed up to 60 minutes with a specific activity of 0.33 pg SP repaired/min/mg SPL. The apparent lag time may result from a need for reduction of SP lyase prior to the initiation of SP repair. ..95 Figure V.4. EPR of SPL used in repair of SP dimers shows that 54% of the protein is in the [4Fe-4S]1+ state, as measured by spin quantification versus a Cu-EDTA standard, prior to its assay for DNA repair activity. ............................. 96 Figure v.5. Time course of SP repair by reduced SPL. A. Representative HPLC chromatograms show loss of the SP peak as a function of time. B. Linear repair is observed up to 50 minutes with a specific activity of 1.33 pmol SP repaired/min/mg SPL. ......................................................................................... 97 Figure Vl.1. HPLC analysis of SAM cleavage to 5’-deoxyadenosine by SPL. HPLC chromatograms of (A) standard sample containing SAM (2.5min) and 5’- deoxyadenosine (8min); (B) control sample containing SAM under assay conditions with no SPL; and assay mixes containing SPL after 90 min (C) and overnight (D). Samples B-D contained 3 mM sodium dithionite, 4 mM D'I'I‘, 30 mM KCI and 25 mM Tris-acetate pH 7.0. Samples C and D also contained 200 pg SP containing pUC18 DNA, 36 pM SAM and 36 pM SPL ............................ 112 Figure VI.2. 3H transfer from labeled [2, 5’, 8 — 3H]-SAM to repaired thymine. DNA repaired by SP lyase in the presence of [2, 5’, 8 - 3H]-SAM (solid lines are duplicate experiments) shows 3H incorporation into thymine (elution time of 3 min.). Samples prepared in the absence of SPL shows no such incorporation (dashed lines). .................................................................................................. 1 15 Figure Vl.3. Tritium isotope effect for H atom abstraction. The bar graph above shows the number of counts present in purified SAM after use in a SP repair reaction with [C-6 - 3H]-thymine SP. Reaction times shown are for 0, 2.5, 5 and 10 minutes. An isotope affect between 15.1 and 17.2 can be calculated for tritium during SP repair and H atom abstraction. ......................................................... 118 xviii Figure VII.1. Anaerobically isolated SPL binding to a 94 base pair DNA oligomer. An increasing amount of SPL was added ranging from 0 pmol (lane 1) to 46 pmol (lane 11), as detailed in Table Vll.1. As more SPL is added to the DNA oligomer, the DNA band is retarded an increasing amount. A dissociation constant of 450 :I: 100 nM can be calculated by measuring the band densities ............................. 132 Figure VII.2. Plot of [SPL-total] versus v ([SPL—DNA]I[DNA]) for the anaerobically as isolated SPL binding to a 94 base pair DNA oligomer .................................. 133 Figure VII.3. Anaerobically isolated SPL exposed to oxygen binding to a 94 base pair DNA oligomer. An increasing amount of SPL was added ranging from 0 pmol (lane 1) to 92 pmol (lane 11), as detailed in Table Vll.2. As more SPL is added to the DNA oligomer, the DNA band is retarded an increasing amount. A dissociation constant of 700 :l: 100 nM can be calculated by measuring the band densities ............................................................................................................ 134 Figure VII.4. Plot of [SPL-total] versus v ([SPL-DNA]/[DNA]) for the apo-SPL binding to a 94 base pair DNA oligomer. .......................................................... 135 Figure VII.5. Apo-SPL binding to a 94 base pair DNA oligomer. An increasing amount of SPL was added ranging from 0 nmol (lane 1) to 1.65 nmol (lane 11), as detailed in Table VII.3. As more SPL is added to the DNA oligomer, the DNA band is retarded an increasing amount. A dissociation constant of 550 :I: 100 nM can be calculated by measuring the band densities .......................................... 136 Figure VII.6. Plot of [SPL-total] versus v ([SPL-DNA]/[DNA]) for the apo-SPL binding to a 94 base pair DNA oligomer. .......................................................... 137 Figure VII.7. Anaerobically isolated SPL with SAM binding to a 94 base pair DNA oligomer. An increasing amount of SPL was added ranging from 0 pmol (lane 1) to 27.6 pmol (lane 11), as detailed in Table VII.4. As more SPL is added to the DNA oligomer, the DNA band is retarded an increasing amount. A dissociation constant of 550 :l: 100 nM can be calculated by measuring the band densities.139 Figure Vll.8. Plot of [SPL-total] versus v ([SPL-DNA]/[DNA]) for the anaerobically as isolated SPL with SAM binding to 94 base pair DNA oligomer ..................... 140 Figure Vlll.1. 35 GHz CW EPR absorbance spectra of SPL in the absence and presence of SAM A. Reduced SPL. B. Reduced SPL with non-labeled SAM. C—F. Reduced SPL with labeled SAM, (c.) 17o SAM; (0.) 15N SAM; (E.) methyl ‘30 SAM; (F.) methyl 2H SAM. ................................................................................ 152 Figure Vlll.2. 35 GHz CW EPR derivative spectra of SPL in the absence and presence of SAM A. Reduced SPL. B. Reduced SPL with non-labeled SAM. C-F. Reduced SPL with labeled SAM, (o) 17o SAM; (o.) 15N SAM; (5.) methyl ‘30 SAM; (F.) methyl 2H SAM. ................................................................................ 153 xix LIST OF SCHEMES Scheme M. An pair of adjacent thymine bases (center) can undergo conversion to a variety of photoproducts when exposed to UV light at 254nm including, a cis, syn cyclobutane dimer (t-s T<>T, upper left), a spore photoproduct (lower left), and a 6-4 photoproduct (6-4 TT, upper right) which can undergo further conversion to a dewar photoproduct (dewar TT, lower right). ............................... 3 Scheme l.2. The UV induced spore photoproduct is repaired directly by Spore photoproduct lyase. The methyl bridge of SP is cleaved by SPL reverting the thymine dimer back to two adjacent thymines ....................................................... 9 Scheme L3. The reactions catalyzed by six members of the radical SAM superfamily are Shown above, illustrating the superfamily’s diverse chemistry... 12 Scheme I.4. Structural comparison of adenosylcobalamin (left) and S-adenosyl- methionine (right) illustrating Similarity of 00-0 and C-8 bond in the two molecules ............................................................................................................ 13 Scheme l.5. Cleavage of SAM by a radical SAM superfamily enzyme yields an adenosyl radical and methionine. ....................................................................... 14 Scheme I.6. ENDOR ENDOR based model of SAM binding to the [4Fe-4S] cluster of PFL-AE. The unique iron is coordinated by the amino nitrogen and carboxyl oxygen. Evidence for orbital overiap between the sutfonium sulfur and the cluster was also obtained. ............................................................................. 16 Scheme I.7. Proposed mechanism for the repair of spore photoproduct by spore photoproduct lyase, involving the cleavage of SAM with subsequent hydrogen extraction from C-6 creating a ring based radical. This C-6 radical abstracts a hydrogen from the methyl bridge resulting in thymine separation. The resulting radical is reabstracted by 5’-deoxyadenosine to reform the adenosyl radical. ....21 Scheme IV.1. Generalized reaction scheme for the enzymatic synthesis of AdoMet. .............................................................................................................. 75 Scheme Vl.1. Proposed mechanism for SP repair by SPL in which SAM is homolytically cleaved by an electron from the iron sulfur cluster of SPL. A putative 5’—deoxyadenosyl radical is formed which abstracts a hydrogen from the C-6 on the thymine ring and initiates radical catalysis. ..................................... 103 Scheme V|.2. S-adenosylmethionine with a tritium label at the 5’-C. ............... 105 Scheme Vl.3. Spore photoproduct label with tritium at C-6. ............................. 105 XV LIST OF ABBREVIATIONS 5’dAdo ....................................................................................... 5’-deoxyadenosine AdoCbl ..................................................................................... adenosylcobalamin AdoMet ........................................................................... S-adenosyl-L-methionine anRNR ............................................................ anaerobic ribonucleotide reductase ATP .................................................................................... adenosine triphosphate APS ...................................................................................... ammonium persulfate B. subtilus ....................................................................................... Bacillus subtilis BioB ............................................................................................... Biotin Synthase B-ME ......................................................................................... B-mercaptoethanol BSA ...................................................................................... bovine serum albumin CPD .................................................................................. cyclobutane photodimer CPM ........................................................................................... counts per minute DFT ....................................... .......................................... density functional theory DNA ...................................................................................... deoxyribonucleic acid DPM ............................................................................... disintegrations per minute D'l‘l' ...................................................................................................... dithiothreitol EDTA .......................... Ethylenedinitrolo)tetra-acetic acid disodium salt dehydrate ENDOR ............................................................ electron nuclear double resonance EPR ................................................................... electron paramagnetic resonance EXAFS ..................................................... extended x-ray absorbtion fine structure FAD ............................................. flavin adenine dinucleotide FPLC ................................................................. fast protein liquid chromatography XX hemN ................................... oxygen independent coproporphyrinogen-lIl-oxidase HPLC ...................................................... high performance liquid chromatography E. coli .............................................................................................. Escherichia coli iMAC .................................................... immobilized metal affinity chromatography IPTG ................................................................ isopropyl-B-D—thioglactopyranoside LAM ................................................................................ lysine 2,3 —aminomutase LB ...................................................................................................... Luria-Bertani LMCT ...................................................................... ligand to metal charge transfer NMR ........................................................................... nuclear magnetic resonance MM ................................................................................................... minimal media MOPS ........................................................ 3-(N-morpholino) propanesulfonic acid PFL ..................................................................................... pyruvate forrnate lyase PFL-AE ................................................ pyruvate forrnate lyase activating enzyme PMSF ....................................................................... phenylmethyl sulfonyl fluoride RNA ............................................................................................... ribonucleic acid SAM ................................................................................ S-adenosyl-L-methionine SDS-PAGE ................. sodium dodecyl sulfate-polyacrylamide gel electrophoresis SP ............................................................................................ spore photoproduct SPL ................................................................................. spore photoproduct lyase SASP .............................................................................. small acid soluble protein T ................................................................................................................. thymine TEMED ....................................................... N,N,N’,N’-tetra-methyI-ethylendiamine TFA ............................................................................................ triflouroacetic acid xxi CHAPTER I INTRODUCTION |.1 Iron Sulfur Clusters in Biology Iron sulfur clusters are among the most versatile and ubiquitous metal- containing structures found in biology. The most obvious and most well known role for these clusters is electron transport, with most types of iron sulfur clusters possessing at least two readily accessible redox states. The flexible protein environment surrounding the cluster creates a wide range of redox potentials in different proteins. This role as an electron transporter is most notably found in ferredoxins, the mitochondrial electron transport chain, rubredoxin, and the proteins of photosynthesis.1' 2 There are three types of iron sulfur clusters found commonly in biology in addition to numerous modifications such as the active site centers in hydrogenase and nitrogenase.3’ 4 The three common cluster types are Shown in Figure M are the [2Fe-28]2+/1+, [3Fe—4S]1+/0 and the [4Fe-4S]3+/ 2” 1+ clusters.1' 2 While most of the initial work on iron-sulfur clusters focused on their roles in electron transport, a number of other roles have emerged for these clusters reflecting their diverse and elegant chemistry. For example, iron sulfur clusters function in regulatory roles,5 turning gene expression on or off in response to the level of iron (the iron-responsive element-binding protein),6’ 7 oxygen (the L. L. 9 9 — Fe—S 59 /S / I I L/l’h, /S\ “‘\\\\\ S SFe Ftle' S S—Fe (Fe\ /Fe'\ / I/ l/ ”a / / ’ L S L [29—3 Fe—S L L/ L/ [4Fe-4S] I3Fe-4S] [ZR-23] Figure L1. The three major categories of iron sulfur clusters are illustrated including the [4Fe-4S], [3Fe-4S], [2Fe-2S] from left to right. 8-10 11-13 fumarate-nitrate reduction protein), and superoxide (SoxR). Iron-sulfur clusters have also been implicated in both redox (nitrogenase,14 carbon monoxide dehydrogenase,15 hydrogenase”) and non-redox (aconitase) catalysis.17 In addition there is evidence of a purely structural role for the cluster I18 in several enzymes including the DNA repair enzymes endonuclease II and MutY.19 One of the most intriguing new roles for iron sulfur clusters has been their involvement in the initiation of radical catalysis in the radical SAM 20—27 superfamily. This role will be discussed in greater detail later. Spore photoprod uct Dewar ‘l'l' Scheme M. An pair of adjacent thymine bases (center) can undergo conversion to a variety of photoproducts when exposed to UV light at 254nm including, a cis, syn cyclobutane dimer (t-s T<>T, upper left), a spore photoproduct (lower left), and a 6-4 photoproduct (6-4 TT, upper right) which can undergo further conversion to a dewar photoproduct (dewar TT, lower right). l.2 DNA Damage, Spore Photoproduct and Small, Acid Soluble Protein Recognized as the inforrnationally active component of almost all genetic material, deoxyribonucleic acid (DNA) was first thought to be an extremely stable macromolecule capable of passing the information required for life from generation to generation with little change.28 It is now know that DNA is not as stable as once thought and is, in fact, subject to constant mutation and damage. These mutations can occur during the replication or recombination of the DNA, from the inherent instability of specific chemical bonds or from environmental factors including chemical (cross-linking agents) and physical (ionizing or UV radiation) sources.28 Specific types of DNA damage include mismatched base pairs during replication, strand breakage by reactive oxygen species, deamination, depurination, and interstrand crosslinking by a variety of chemical agents.28 Whatever form of DNA damage that occurs, all of them are harmful to cell viability. Without repair, DNA replication as well as gene transcription will be blocked, inhibiting the cell from producing the proteins and enzymes necessary for cellular function. As noted above, DNA can be damaged in a number of ways; one such example is by exposure to ultraviolet (UV) light. It has been shown that DNA exposed to ultraviolet light contains a number of damaged DNA photoproducts involving the pyrimidine bases.29 30 Among these photoproducts, the primary type of damage is the cyclobutane dimer (CPD), shown in Scheme l.1. However, secondary photoproducts are also produced such as 6-4 photoproducts.29 In the mid 1960’s, an intriguing phenomenon was discovered among sporulating bacteria such as Bacillus and Clostridium. When exposed to large amounts of ultraviolet light, most types of bacteria cannot survive; Bacillus and Clostridium spore were however shown to have a high resistance towards UV light, being 5 to 50 times more resistant to UV light than normal vegetative cells.31 A clue to this increased resistance was found by examining the type of DNA damage in UV exposed spores. It was discovered that the major photoproduct in irradiated spores was not cyclobutane dimers, as found in vegetative cells, but 5-thyminyl- 5,6-dihydrothymine (now known as Spore photoproduct, SP) (Scheme l.1). 29' 32 In addition to the different type of DNA damage incurred, the protein composition of bacterial spores is unique. Up to 20% of protein in the Spores of various Bacillus species is a group of proteins called small, acid soluble protein (SASP).33 The small, acid soluble proteins are so named because the proteins were found to be soluble in acidic mixtures and thus could be dissolved in acidic solutions while other proteins precipitated, allowing for easier purification. These proteins range from 60-73 amino acids (5-7kDa) and appear approximately 3-4 hours after the onset of Sporulation.33 The SASPS can be divided into two types, the y and the a/B SASPS.33 Significant properties of the y-type SASPS include: 1) They are encoded by a single gene in all species. 2) Their amino acid sequences are highly conserved but not as highly as the all} SASP. 3) They are not associated with spore DNA in vivo.34 4) Their only known role is as an amino acid reserve for spores during germination. Characteristics of the a/B-type SASP include: 1) They are encoded by a family of seven genes. 2) Their amino acid sequence is highly conserved across families. 3) They are associated with spore DNA in vivo.34 4) They play a major role in determining the properties of spore DNA in vivo and resistance of spores to UV light.” During spore germination, the SASPS are rapidly degraded to amino acids with their degradation initiated by a novel endonuclease termed the germination protease.36 The presence of the ct/B-type SASP is associated with the spores UV resistance and spores that contain deletion mutants for SASPS show a markedly lower UV resistance.37 In vivo studies have shown that ct/B-type SASPS are associated with the spore’s chromosomal DNA and this may protect the DNA 38,39 from damage. It has been proposed that the SASP promotes a 40.41.42 conformational change in the DNA when it binds. Cells in the vegetative state are found in the B conformation while spectroscopic evidence suggests the DNA in spores is best described as being in an A conformation. The A conformation of DNA has more bases per helix turn and a wider diameter than the B conformation, which may promote the formation of the methylene bridge in SP as opposed to the cyclobutane dimer.”42 SASP has also been shown to bind DNA in vitro and overexpression in E. coli. can partially change the properties of a cell to be more Spore like.43 However, the mechanism by which SASP promotes the conformational change in DNA or why this causes the major photoproduct to be changed from the cyclobutane dimer to the spore photoproduct remains unclear. l.3 Spore Photoproduct Lyase Damage to a cell’s DNA is extremely harmful and can lead to mutations in enzymes crucial for cellular function, causing reduced cell viability and increased cell apoptosis.28 It is therefore important that a cell has a method for repairing this damage either by a direct reversal pathway or by nucleotide excision repair. In nucleotide excision repair the specific base and those around it are cutout of the DNA strand and replaced by new base pairs leaving the strand repaired.“ 45 The direct reversal pathway on the other hand requires an enzyme to Specifically repair the one or more bases that are damaged. An example of this occurs with the aforementioned cyclobutane dimer. An enzyme known as DNA photolyase utilizes light to cleave the two bonds forming the thymine bridge.46 This light driven enzyme contains two cofactors; one is always a flavin-adenine dinucleotide (FAD) and the other is a folate, methenyltetrahydrofolate (MTHF) or a deazaflavin, 8-hydroxy-7, 8-didemethyl-5—deazariboflavin (8-HDF).47 Accordingly, the enzymes have been classified into folate class and deazaflavin class photolyases. The reaction occurs by the DNA photolyase binding to the damaged DNA and flipping the CPD out of the helix and into the active site cavity 46. 47 of DNA photolyase. The folate then absorbs a blue light photon and transfers the excitation energy to the flavin, which then transfers an electron to the CPD; the 5-5 and 6-6 bonds of the cyclobutane ring are now in violation of Huckel rules, and therefore, the CPD is Split to form two pyrimidines.“ 47 Concomitantly, an electron is transferred back to the nascently formed FADH to regenerate the FADH' form. DNA photolyase is one of the most studied DNA repair proteins; when the initial spore photoproduct was discovered, it was hypothesized that a novel type of DNA photolyase might exist in sporulating bacteria utilizing a similar pathway 47, 48 to cleave the spore photoproduct. Sequence homology studies found that there was gene (splB) that showed some C-tenninal homology to the DNA 49, 50 photolyases (Table I.1). Another study of this gene found that deletion mutants did not have as high of survival capabilities when exposed to UV Iight.48' 51 . N.c. WSYNVDHFHAWTQGRTQFgIIDAAMRQVLSTQYMHNRLRMI 477 . s.c. WENNPVAFEKWCTGNTQIEIVDAIMRKLLYTQYINNRSRMI 454 - E.c. WQSNPAHLQfiMQEGKTgngVDAAMRQLNSTQQNHNRLRMI 346 . H.h. WRD§PAALQ§WSDGET§§§IVDAGMRQLRAEAYMHNRVRMI 353 - A.n. WENREALFT§WTQAQT§X§IVDAAMRQLTETgwMHNRCRMI 353 - S.g. WRsfiADEMHéwfisGLTQffiLVDAAMRQLAfiEqfiMHNRARML 334 . B. s . sPLQKRIEA§V§VAKA¢¥§LGFIVAPIYIHEGWEEGYRHLF 2 50 Table I.1. C-tenninal sequence homology between DNA photolyases from . different organisms (above) and spore photoproduct lyase from Bacillus subtilis (below) shows several conserved residues. sugar—N 0 sugar—-N O / uv / H H CH3 T phosphate phosphate 0 H \ O H I SPL I )—~ M sugar—N >=0 sugar——N O H CH3 H H CH3 Scheme l.2. The UV induced spore photoproduct is repaired directly by spore photoproduct lyase. The methyl bridge of SP is cleaved by SPL reverting the thymine dimer back to two adjacent thymines. Dark repair of SP with the new enzyme spore photoproduct lyase (SPL, Scheme l.2) was later demonstrated, differentiating it from the DNA photolyases by functioning independent of light.45 If SPL does not utilize light to carry out DNA repair, then it must have a different and novel repair mechanism. In fact, sequence homology studies show that it contains a specific sequence, CXXXCXXC (Table l.2), which places it with a group of enzymes known as the radical SAM superfamily.52 Work carried out by the Nicholson lab confirmed that like other members of the radical SAM superfamily SPL requires the presence of S-adenosylmethionine (SAM) and contains an iron-sulfur cluster. Unlike the DNA photolyases, no flavin is present as a cofactor and no light is needed to initiate DNA repair.52' 53 SP Lyase 86 IPFATGEMGHQHYéYLQTT . PFL—AE 24 ITFFQGCLMRCLYCHNRDT . aRNR-AE 20 VLFVTGQLHKCEGQYNRST - Biotin Synthase 47 SIKTGAoPQDcKYcPQTSR . Lipoate Synthase 48 MILGAICTRRCPFCDVAHG . LAM 132 LLITDMCSMYCRHCTRRRF . HemN 53 YFHIPFcQSMcLYoGCSIH - ThiH 90 LYLSNYCNSKCVYCGFQIL - MoaA 15 IAVTPECNLDCFFCHMEFK . BssD 68 TIFLKGQNYKgGFgFHTIN Table l.2 CX3CX2C conserved binding motif of the radical SAM superfamily. I.4 Radical SAM Superfamily The radical SAM superfamily is composed of a wide range of proteins with different functionalities and is thought to be composed of over 600 different enzymes.20 These enzymes range in function from the sulfur insertions 54, 55 of biotin synthase (BioB) and lipoate synthase (LipA)56 to the protein radical 57, 58 activation of pyruvate formate lyase activating enzyme (PFL-AE) , anaerobic 59, 60 ribonucleotide reductase activating enzyme (anRNR), and benzylsuccinate synthase activating enzyme.61 The superfamily contains enzymes that catalyze rearrangement reactions such as lysine 2,3-aminomutase (LAM)62 and proteins like oxygen-independent coproporphyrinogen-lll oxidase (HemN) involved in heme biosynthesis.63 More recently they have been implicated in the formation of 64, 65 iron sulfur clusters in hydE and hde and the formation of molybdoterin by moaA.66 Given the diversity of reactions catalyzed above, it is then not 10 surprising that the radical SAM superfamily would contain an enzyme capable of 52.53 DNA repair such as spore photoproduct lyase. The diversity of the superfamily is summarized in Scheme L3 and illustrates the wide ranging chemistry of its members. The radical SAM superfamily is a composed of a group of protein with several unique characteristics. First, all members of the radical SAM superfamily share a cysteine motif, CX;CXZC. (Table I2)”25 This amino acid sequence coordinates an iron-sulfur cluster as demonstrated by several members of the 62.67.68 superfamily. Among proteins with iron sulfur clusters, this sequence is unique because it only has 3 cysteinal ligands capable of coordinating the SKFZS cluster. This leaves open the possibility of either a [3Fe-48] cluster or a [4Fe- 4S] cluster with one non-cysteinal ligand being present. In addition to having the conserved sequence motif, all members in the family coordinate an iron sulfur cluster and all members of the family utilize the small molecule S-adenosyl-L- methionine.”25 S-Adenosyl-L-methionine is a naturally occurring molecule distributed in virtually all body tissues and fluids. It is of fundamental importance in a number of biochemical reactions involving enzymatic transmethylation, contributing to the synthesis, activation and/or metabolism of such compounds as hormones, neurotransmitters, nucleic acids, proteins, phospholipids and certain drugs.69 Its most prominent role has been that of a methyl donor involved in reactions such as the methylation of DNA and RNA.69 Its role in the radical SAM superfamily is both new and unexpected.”25 ll it . HN NH A HN NH O O O S . o +H3Hp‘, coo- Biotin synthase Lysine 2,3 aminomutase .5 S /swmn—N sugar—N Koa/ 7—2—0 > phosp/hate H2 phosph/ate \ )—N \ sugar—N sugar—NW O O H H CH3 Lipoyl Synthase Spore photoproduct lyase COOH HOOC Oxygen-independent Activating Enzymes coproporphyrinogen-lll oxidase Scheme L3. The reactions catalyzed by six members of the radical SAM superfamily are shown above, illustrating the superfamily’s diverse chemistry. 12 The discovery of the radical SAM superfamily started in the late 1960’s and early 1970’s with work carried out by Barker and coworkers on the isomerase, lysine 2,3 aminomutase, which was shown to require SAM rather then the cofactor, adenosylcobalamin (AdoCbl),used by many other isomerases(Scheme l.4).70 AdoCbl dependent enzymes function by cleaving Scheme l.4. Structural comparison of adenosylcobalamin (left) and S-adenosyl- methionine (light) illustrating similarity of 00-0 and C-S bond in the two molecules. the 00-0 bond to form an adenosyl radical that can initiate radical catalysis.71 Later work carried out by Knappe and coworkers on pyruvate formate lyase (PFL) showed that it required a second enzyme for activity; this second enzyme was a SAM-dependent activase which generated a glycyl radical.72 Given the above information. it was thought that the members of the radical SAM superfamily cleave SAM at the C-S bond to form methionine and a 5’deoxyadenosyl radical intermediate (Scheme IS), the latter being the same intermediate implicated in the AdoCbl enzymes.”26 -OOC NH3+ SAM methionine Scheme I.5 Cleavage of SAM by a radical SAM superfamily enzyme yields an adenosyl radical and methionine. When the first members of the radical SAM superfamily were being characterized, a review on LAM was published that called S’adenosylmethionine, “a poor man’s adenosylcobalamin,”73 mainly because of the relatively simple structure of AdoMet when compared to AdoCbl and because the C-S bond, with 14 an energy of 60 kCal, is double the energy of the C-Co bond at 60 kCal. However, the diversity and complexity of the chemistry carried out in the radical SAM superfamily caused a change in thinking a few years later when a new review by the same author, switched the name to “a wolf in sheep’s clothing or a 9:23 rich man’s adenosylcobalamin. While there is still no direct Spectroscopic evidence for the presence of this adenosyl radical in the radical SAM reactions, work carried out on LAM allowed direct detection of an allylic analog of the putative 5’-deoxyadenosine radical intermediate."' 75 As previously mentioned, with only three cysteine residues available to coordinate the iron sulfur cluster, the potential exists for multiple kinds of iron sulfur clusters to be present, along with the possibility of a non-cysteinal fourth ligand to coordinate a [4Fe-4S] cluster. Initial isolation and characterization of several enzymes including PFL-AE and BioB showed several cluster states present in the protein, including a [3Fe-4S]1+ and [4Fe-4S]2+ that could be reduced to a [4Fe-4S]1+.68' 76' 77 An elegant study by Broderick and co-workers with electron paramagnetic resonance (EPR) spectroscopy correlated the disappearance of the [4Fe-4S]1+ signal with the appearance of an organic glycyl radical signal.78 Other studies on LAM and anRNR had also shown the presence of a [4Fe-4S]1+ Signal in active protein.6°' 79 Taken together these results strongly suggested that the active cluster of the radical SAM superfamily was the [4Fe- 4S]1+ cluster. If the active cluster of the superfamily is the [4Fe-4S]1+, then with 15 only 3 cysteinal ligands present to coordinate the cluster, the cluster is expected to be site—d ifferentiated. Another well studied enzyme that contains a unique iron is aconitase. Electron double nuclear resonance (ENDOR) studies on aconitase confirm coordination to the unique iron site by the substrate citrate via the carboxyl oxygens.8°‘82 Model structures based on ENDOR studies in coordination with Mdssbauer spectroscopy on PFL-AE confirmed the presence of the unique iron in the radical SAM superfamily.58' 83' 84 The data provided evidence for coordination to the unique iron by the amino nitrogen and carboxyl oxygen of SAM, analogous to the coordination of citrate to aconitase (Scheme l.6).84 Scheme I.6. ENDOR based model of SAM binding to the [4Fe-48] cluster of PFL-AE. The unique iron is coordinated by the amino nitrogen and carboxyl oxygen. Evidence for orbital overlap between the sulfonium sulfur and the cluster was also obtained. 16 ENDOR experiments also show an orbital overlap between the cluster and the sulfonium from SAM. Extended x-ray absorption fine structure (EXAFS) studies on the PFL-AE/Se-SAM interaction Show no interaction between the selenium cation and the unique iron of the cluster, suggesting that the orbital overlap occurs via a bridging sulfide.85 Together, the results suggest the possibility of sulfur centered rather than iron-centered, redox chemistry.83 ENDOR and EXAFS studies carried out on LAM, however, suggest Slightly different model. While the ENDOR studies of LAM also show coordination to the unique iron as with PFL-AE, the sulfonium appears to interact with the iron of the cluster, suggesting a slightly different binding mode and 86‘ 87 Both of these results suggested an possibly iron centered redox chemistry. inner sphere pathway for electron transfer in the homolytic scission of the 5’ C—S bond in SAM.57’ 58' 83' 86' 87 Noteworthy to this discussion, PFL-AE utilizes SAM as a substrate, while in LAM it acts as a cofactor. This is a possible rationale for the difference of sulfonium interaction in PFL-AE and LAM. With the recent explosion of interest in the radical SAM superfamily several crystal structures have been reported including HemN, BioB, MoaA and LAM.63' 66’ 88’ 89 A striking feature of these crystal structures is their similarity to each other despite the diversity of their chemistry being performed. All of the structures solved to date, share a (W006 repeat motif in their structural cores. Alignment of this motif for all four structures places the iron—sulfur cluster at the same location relative to the motif. The B-sheet formed by this motif is extended 17 on both sides in MoaA, HemN, and LAM to form a larger crescent channel closed by an additional domain, whereas the sheet is extended by two additional B/or repeats in BioB which closes to form a TIM barrel.89 The location of SAM relative to the iron sulfur cluster is also confirmed in these crystal structures, with the binding of the unique iron by the amino nitrogen and carboxyl oxygen.63' 66’ 88"” l.5 Recent Work on Spore Photoproduct Lyase Initial work on SPL in the Nicholson lab showed conclusive evidence for SPL’S ability to repair spore photoproduct in vitro, with Specific binding to SP and not CPD.91 In addition to this work, it was shown that SPL required SAM to carry out DNA repair and did this in the absence of light.52 Further work on SPL confirmed the presence of an iron sulfur cluster in the protein, with the as- isolated protein containing a [3Fe-4S]1+ that can be reduced with dithionite to a [4Fe-4S]1+ cluster as monitored by EPR.53 High performance liquid chromatography (HPLC) studies provided evidence for SAM cleavage by the enzyme with loss of the [4Fe-4S]1+ EPR signal.53 The same study proposed SPL to be a homodimer with the iron sulfur cluster serving to bind the two subunits.53 Based on recent crystal structure of four other radical SAM superfamily members, the proposal of a homodimer with the cluster at the center seems unlikely. It is also noteworthy that the experiments in this publication did not involve protein that was shown to be active in SP repair.53 18 A mechanism for SPL was published in 1999 by Mehl and Begley in which SAM is utilized as a protein cofactor (Scheme l.7).92 In this mechanism an electron from the cluster is transferred to SAM whereupon SAM is cleaved to methionine and the putative 5’-deoxyadnosyl radical. This radical abstracts hydrogen from the C-6 position creating a carbon radical on the thymine ring which induces a radical mediated B-scission. The subsequent carbon radical reabstracts a hydrogen atom from 5’-deoxyadenosine and reforms the initial adenosyl radical. This can then go on to further catalysis or reform SAM by the loss of an electron to the cluster.92 This mechanism iS supported by the work of Cheek and Broderick in which SP tritiated at the C-6 position is transferred to SAM, where as SP tritiated at the methyl bridge is not seen in SAM.93 The mechanism is further confirmed by a Hartree-Fock/density functional theory calculation (DFT) with a slight modification where an additional inter-thymine hydrogen transfer step taking place before the reabstraction of hydrogen to the 5’-deoxyadenosine. The DFT calculations Show the last step to be rate determining.94 It has been Shown more recently that UV irradiation of DNA can produce both inter-strand and intra-strand spore photoproducts.‘“i'5'97 A recent study has concluded that SPL can repair the inter-strand but not the intra-strand spore photoproduct. In this study, SP was synthetically produced without incorporation into a DNA strand and repaired with SPL. Repair was tracked via HPLC and mass spectrometry.98 This is an intriguing result but without incorporation of the synthetic SP into a DNA strand it is difficult to apply it to in vivo conditions. 19 Scheme l.7 Proposed mechanism for the repair of spore photoproduct by spore photoproduct lyase, involving the cleavage of SAM with subsequent hydrogen extraction from C-6 creating a ring based radical. This C-6 radical abstracts a hydrogen from the methyl bridge resulting in thymine separation. The resulting radical is reabstracted by 5’-deoxyadenosine to reform the adenosyl radical. 20 H 3_N,HO sugar—N O sugar—N / H e- H phosphate H phosphate H \ ,HC 2 _L__.\ o j-IC 2 3—N )—N sugar—N 0 sugar—N O H H CH3 H H CH3 |=:l H (EH? H CH3 p.“ O A +S : O A g -ooc—(V 000W HO ii NH3+ HO OH NH3+ TT SP 0 H o H >~—N )—N SU987—-N O sugar—N __ 0 phosphate H CH3 phosphate H CH2 \ o .H ) SUQBF-N NH sugar—N o H H CH3 H ' CH3 .54 H CH . H = A 1" H (EH3 w -oocW SEH 3 HO 3 NH3+ -OOC4(\/ NH3+ sugar——N?_ NH O sugar—N phosphate H CH3 phosphate 0 ,H \ . )—-N sugar —N O <—— sugar—N _ ?= H CH3 ills <.= ”(VS S -OOC~(\/ -OOCW NH3+ NH3+ Scheme 1.7 21 The results reported within, further add to the knowledge base on the spore photoproduct lyase with a focus on cluster identification, SAM cluster interaction, SP repair kinetics and SPL-DNA interaction. We have purified SPL with a higher iron and sulfur content than previously reported and shown under these conditions in the absence of cluster reconstitution that it is monomeric. Our study further analyzes the cluster with an array of spectroscopic techniques and looks at the enzyme mechanism with the conclusion that unlike, PFL-AE but similar to LAM, SAM is utilized as a cofactor by SPL. 22 LB 10. 11. References Beinert, H., Iron-sulfur proteins: ancient structures, still full of surprises. J. Biol. Inorg. Chem. 2000, 5, 2-15. Beinert, H.; Holm, R. 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M., Oxygen-17, proton, and deuterium electron nuclear double resonance characterization of solvent, substrate, and inhibitor binding to the iron-sulfur [4Fe-4S]+ cluster of aconitase. Biochemistry 1990, 29, (46), 10526-32. Werst, M. M.; Kennedy, M. C.; Houseman, A. L. P.; Beinert, H.; Hoffman, B. M., Characterization of the iron-sulfur [4Fe-4S]+ cluster at the active site of aconitase by iron-57, sulfur-33, and nitrogen-14 electron nuclear double resonance spectroscopy. Biochemistry 1990, 29, (46), 10533-40. Walsby, C. J.; Hang, W.; Broderick, W. E.; Cheek, J.; Ortillo, D.; Broderick, J. B.; Hoffman, B. M., Electron-Nuclear Double Resonance Spectroscopic Evidence That S-Adenosylmethionine Binds in Contact with the Catalytically Active [4Fe-4S]+ Cluster of Pyruvate Formate-Lyase Activating Enzyme. Journal of the American Chemical Society 2002, 124, (12), 3143-3151. Walsby, C. J.; Ortillo, D.; Broderick, W. E.; Broderick, J. B.; Hoffman, B. M., An Anchoring Role for FeS Clusters: Chelation of the Amino Acid Moiety of S-Adenosylmethionine to the Unique Iron Site of the [4Fe-48] Cluster of Pyruvate Formate-Lyase Activating Enzyme. Journal of the American Chemical Society 2002, 124, (38), 1 1270-1 1271 . Casper, M. M.; Casper, N. J.; Hang, W.; Shakes, J. E.; Broderick, W. E.; Broderick, J. B.; Johnson, M. K.; Scott, R. A., Sthctural studies of the interaction of S-adenosylmethionine with the [4Fe-4S] clusters in biotin synthase and pyruvate formate-lyase activating enzyme. Protein Science 2003, 12, (7), 1573-1577. Chen, D.; Walsby, C.; Hoffman, B. M.; Frey, P. A., Coordination and Mechanism of Reversible Cleavage of S-Adenosylmethionine by the [4Fe- 481 Center in Lysine 2,3-Aminomutase. Journal of the American Chemical Society 2003, 125, (39), 1 1788-11789. Casper, N. J.; Booker, S. J.; Ruzicka, F.; Frey, P. A.; Scott, R. A., Direct FeS Cluster Involvement in Generation of a Radical in Lysine 2,3- Aminomutase. Biochemistry 2000, 39, (51 ), 1 5668-15673. Berkovitch, F.; Nicolet, Y.; Wan, J. T.; Jarrett, J. T.; Drennan, C. L., Crystal structure of biotin synthase, an S-adenosylmethionine-dependent radical enzyme. Science 2004, 303, (5654), 76-80. Lepore, B. W.; Ruzicka, F. J.; Frey, P. A.; Ringe, D., The x-ray crystal structure of lysine-2,3-aminomutase from Clostridium subterrninale. Proceedings of the National Academy of Sciences of the United States of America 2005, 102, (39), 13819-13824. 30 90. 91. 92. 93. 94. 95. 96. 97. 98. Layer, G.; Kervio, E.; Morlock, G.; Heinz, D. W.; Jahn, D.; Retey, J.; Schubert, W.-D., Structural and functional comparison of HemN to other radical SAM enzymes. Biological Chemistry 2005, 386, (10), 971-980. Slieman, T. A.; Rebeil, R.; Nicholson, W. L., Spore photoproduct (SP) lyase from Bacillus subtilis specifically binds to and cleaves SP (5- thyminyl-5,6-dihydrothymine) but not cyclobutane pyrimidine dimers in UV- irradiated DNA. J. Bacteriol. 2000, 1 82, (22), 641 2-7. Mehl, R. A.; Begley, T. P., Mechanistic Studies on the Repair of a Novel DNA Photolesion: The Spore Photoproduct. Organic Letters 1999, 1, (7), 1065-1066. Cheek, J.; Broderick, J. B., Direct H atom abstraction from spore photoproduct C-6 initiates DNA repair in the reaction catalyzed by spore photoproduct lyase: evidence for a reversibly generated adenosyl Radical lntennediate. Journal of the American Chemical Society 2002, 124, (12), 2860-2861. Guo, J.-D.; Luo, Y.; Himo, F ., DNA repair by spore photoproduct lyase: a density functional theory study. Journal of Physical Chemistry B 2003, 107, (40), 11188-11192. Douki, T.; Setlow, B.; Setlow, P., Effects of the binding of aIb-type small, acid-soluble spore proteins on the photochemistry of DNA in spores of Bacillus subtilis and in vitro. Photochemistry and Phatabialagy 2005, 81, 1 63-169. Douki, T.; Laporte, G.; Cadet, J., Inter-strand photoproducts are produced in high yield within A-DNA exposed to UVC radiation. Nucleic Acids Research 2003, 31, (12), 3134-3142. Douki, T.; Cadet, J., Formation of the spare photoproduct and other dimeric lesions between adjacent pyrimidines in UVC-irradiated dry DNA. Photochemical & Phatabiangical Sciences 2003, 2, (4), 433—436. Friedel, M. G.; Berteau, O.; Pieck, J. C.; Atta, M.; Ollagnier-de-Choudens, S.; Fontecave, M.; Carell, T., The spore photoproduct lyase repairs the SS- and not the 5R-configured spore photoproduct DNA lesion. Chemical Communications 2006, (4), 445-447. 31 CHAPTER II GROWTH AND PURIFICATION OF SPORE PHOTOPRODUCT LYASE ".1 Introduction The advances in the field in molecular biology throughout the 1950’s and 1960’s had a major impact on the study of protein structure and function. Protein purification in the past had been both difficult and time consuming. It required a large amount of cells from the organism being studied, with the protein of interest possibly consisting of less than 1% of the total protein present in the cells. Characterization of human proteins was particularly difficult as it was hard to obtain sufficient quantities of many human organs and cells for study. With such a low percentage of protein present, purification could require well over ten steps, during which time, much of the protein was lost or degraded.1 The discovery and development of plasmid derived cloning vectors greatly improved in vitro studies of proteins. Plasmids are circular molecules of DNA that replicate autonomously in a bacterial host cell. While not essential to cellular survival, plasmids can confer selective advantages such as antibiotic resistance. This made them ideally suited for development as cloning vectors, and since the early 1970’s naturally occurring plasmids have been altered to include among other things, multiple cloning sites, promoters, and lac operators and repressors. 32 With these vectors, the gene of interest can be cloned into the plasmid by use of restriction enzymes and overexpression can be controlled by the presence or absence of an inducer molecule such as isopropyl B-D-thiogalacto-pyranoside (IPTG).2 Despite these advances in protein overexpression and cloning, protein purification could still be difficult. Multiple separation columns were typically needed, along with dialysis and other purification techniques, to achieve a sufficiently pure protein sample. The invention of immobilized metal affinity chromatography (iMAC) in 1975 by Porath was thus a large benefit to the field of biochemistry.3 With this purification technique, a protein is cloned with a specific multiple histidine tag sequence located at either the C-terrninus or N-terrninus of the protein. After cell growth, the cell lysate is purified over a column containing a resin that has a link between the beads and a nitrogen/oxygen binding claw that can coordinate a divalent metal such as nickel, cobalt, iron, manganese or magnesium. Some of the more common commercial resins used are; TALONTM from BD Clontech, nickel-nitrilotriacetic acid (Ni-NTA) from Qiagen, and Ni- Sepharose from GE Healthcare. The histidine tagged protein will coordinate to this metal as it is run over the column, while other proteins generally wash straight through the column. A high concentration of imidazole is then used to wash the column to elute the his-tagged protein.4 This method yields a large quantity of protein free of other contaminants. The iMAC approach has been employed to overexpress and purify the spare photoproduct lyase from Bacillus subtilis with gene insertion into a pET14b 33 expression vector containing an N-terrninal 6 histidine tag. After insertion and transformation into E. coli, metal affinity chromatography is used to purify the protein for further in vitro characterization. ".2 Experimental Methods Materials All chemicals used were commercially obtained except when noted othenrvise and were of the highest purity. Tuner(DE3)pLysS competent cells were purchased from NovagenTM. The Ni/Co-sepharoseTM column was purchased from Amersham Biosciences now GE Healthcare and used interchangeably. SDS- PAGE gels were commercially obtained from Bio-Rad Scientific. Growth of spare photoproduct lyase The spIB gene from Bacillus subtilis used in this work had been previously cloned into the pET14b expression vector from Novagen in our lab by Dr. Jennifer Cheek. A single colony of the overexpression strain pET14b-SPL17 transformed into E. coli Tuner(DE3)pLysS competent cells was used to inoculate 50 mL of Luria/Bertani (Miller’s Modification)5 medium containing 50 pg/mL of ampicillin. This culture was grown to saturation at 37 °C and used to inoculate a 10L flask of minimal media/ampicillin described elsewhere.6 The 10 L culture was grown at 37 °C in a New Brunswick Scientific fennentor (250 rpm, 5 Umin Oz). When the culture reached an 00600 = 0.6, isopropyl B-D-thiogalacto- pyranoside (IPTG) was added to 1 mM final concentration and the medium was 34 supplemented with 750 mg Fe(NH4)2(SO4)2 . The culture was grown for an additional 2 hours, and then was cooled to 25 °C and placed under nitrogen (5 Umin). The culture was further cooled to 15 °C, moved to a 4 °C refrigerator and left under nitrogen overnight for 12 hours. The cells were then harvested by centrifugation at 4°C and stored under nitrogen at -80 °C for further use. Purification of spare photoproduct lyase Spore photoproduct lyase was purified from E. coli Tuner(DE3)pLysS transformed with pET14b-SPL17, prepared as described above. All steps in the purification were performed in a single day under strictly anaerobic conditions in a Coy anaerobic chamber (Coy Laboratories, Grass Lake, MI) at 4 °C except where noted. Solutions and buffers used in the purification were thoroughly degassed by sparging with nitrogen or by repeated pump / purge cycles on a Schlenk line prior to bringing them into the Coy chamber. The pelleted cells (13 to 19 grams) were brought into the anaerobic chamber and resuspended in 20- 30 mL of pH 8.0 lysis buffer containing 20 mM sodium phosphate, 500 mM NaCl, 1% Triton X-100, 5% glycerol, 10 mM MgCl2, 20 mM imidazole, 1 mM PMSF, 1 mg Iysozyme per gram of cells, and 0.2 mg DNAse l and RNAse A. This suspension was agitated for one hour until homogenous and then centrifuged at 27,000 x g for 30 min at 4 °C. The resulting crude extract was loaded directly onto a Ni/Co-SepharaseTM High Performance affinity column (0.7 X 2.5 cm, 1mL) that had been previously equilibrated with buffer A (20 mM sodium phosphate, 500 mM NaCl, 10 mM imidazole, 5% glycerol, pH 8.0). The column was washed 35 with 15 mL of buffer A, and then a 25 mL step gradient (5 mL steps at 10%, 20%, 50%, 70%, 100% buffer B) from buffer A to buffer B (20 mM sodium phosphate, 500 mM NaCI, 500 mM imidazole, 5% glycerol, pH 8.0) was run to elute the adsorbed proteins. SP lyase eluted as a sharp brownish band at 50% buffer B in the step gradient. The fractions were analyzed by SDS-PAGE, and those judged to be 295% pure were pooled and concentrated at 4 °C using an Amicon concentrator equipped with a YM-10 membrane. The protein was placed in o- ring-sealed tubes, flash-frozen, and stored at -80 °C. Protein, iron, and sulfide assays. Routine determinations of protein concentrations were done by the method of Bradford,7 using a kit sold by Bio-Rad and bovine serum albumin as a standard. Iron assays were performed using the methods of Fish or Beinerta’ 9 Sulfide assays were carried out as previously described by Broderick et al.6 using a modification of the method developed by Beinert .10 Gel filtration chromatography SPL (25 uL) was loaded onto a pre-equilibrated (20 mM sodium phosphate, 500 mM NaCl, 5% glycerol, pH 8.0) sepharoseTM 12 column (1 cm x 30 cm, Pharrnacia) at 0.25 mL/min and run with a isocratic flow for 100 min at 1 mL/min using the same buffer. Bio-Rad gel filtration standard was loaded and run under the above conditions to check for molecular weight. The UV absorbance was monitored at both 280 nm and 426 nm. ‘ 36 ".3 Results And Discussion Purification of Spare Photoproduct L yase The SPL expression vector, pET14b-SPL17, was used to transform E. coli Tuner(DE3)pLysS for overproduction of histidine-tagged SPL (SPL-Hise), which migrates at approximately 43 kDa on SDS-PAGE (Figure Il.1). The overexpressed cells were lysed using an enzymatic lysis procedure. The crude extract was then passed through a Co-SepaharoseTM High Performance affinity column (Figure "2), pure fractions (>95 %) were identified by SDS-PAGE (Figure "3), pooled, concentrated, and stored under nitrogen at —80 °C. If the SPL-Hiss was not sufficiently pure after a single run over the affinity column, the protein was dialyzed and re-purified over the same column. Typically approximately 25 mg of pure protein is obtained from 10 L of growth media. SPL elutes as a dark brown band from the Ni/Co-SepaharoseTM High Performance column, consistent with the presence of an iron sulfur cluster in the protein. The anaerobically purified SPL has been found to contain iron (3.1 mol Fe per mol SP lyase and acid-labile sulfide (3.0 :I: 0.3 mol 8'2 per mol SPL). The amount of iron present in the purified SPL is dependent on the precise growth and purification conditions. The purified SPL exhibited instability at higher concentrations and precipitated above ~250 uM. Addition of a 5X excess of SAM to the protein resulted in concentration of upwards of ~750 (M and increased stability at room temperature. This could indicate a stabilizing structural role for SAM in SPL." 37 66 kD was 45 kD ........ 31 kD ------ as 1 2 3 4 Figure ".1 SDS-PAGE of the purification of SPL from the metal affinity column chromatography, standards are shown on the left in lane 1. Lane 2 shows uninduced cells, lane 2 shows cell after addition of 1 mM IPTG to induce the cell culture. Lane 4 shows protein after purification an a Co-sepharose column. 38 3750 ‘ —280 nm - - - 426 nm ‘—“ SPL 2750 ‘ — % Buffer B 3 E 1750 i 100% 750 ‘ 50% 0% .4 ‘ _ " "L ‘ _'_ 1 - - r ‘ ' ' ' ': """"" r' ' ' -250 0 10 20 30 Figure ".2 FPLC chromatogram of SPL purification using cobalt metal affinity chromatography. SPL elutes ~20 min at 50% during an imidazole step gradient as a brownish-red band with a high absorbance at both 280 nm and 426 nm. Subunit Structure of Spare Photoproduct L yase. SPL has been previously reported to be dimeric under conditions favoring reconstitution of iron-sulfur clusters, and to likely contain a subunit-bridging iron sulfur cluster.12 As our SPL is purified with a significant proportion of iron sulfur clusters intact, we have been able to re-investigate the question of subunit structure using protein that has not been subjected to artificial reconstitution. Analytical gel-filtration chromatography shows that SPL migrates 39 45 kDa Figure ".3 SDS-PAGE gel electrophoresis of crude extract and fractions eluting from the Co-sepharose column. Lane 1: Molecular weight standard. Lane 2: Crude Iysate before loading onto iMAC column. Lane 3-5: 1 mL fractions from Co-sepharose columns. with an apparent molecular mass of 46 kDa (Figure |l.4). Simultaneous detection at 426 and 280 nm shows that the visible chromophore elutes with this 46 kDa peak. A small shoulder on this peak (~85 kDa) is visible with both 280 and 426 nm detection and may represent a small amount of SP lyase dimer, which could be an artifact of the high protein concentrations used. These results together with the ironzprotein ratios we obtain for purified SP lyase, provide support for SP lyase as a monomer binding a single [4Fe-4S] cluster.11 40 670kDa 158kDa 44kDa 17kDa I I I I SP Lyase 46kDa —* 2: '5 C .9. E (D .2 E (D (I —280 nM - - - 426 nM I MW Std o ‘ W 0 4 8 12 mL Figure ".4 Gel filtration chromatography of a SPL on a sepharoseTM 12 column. SPL loaded on a gel filtration column and run with isocratic flow shows partially purified SPL elutes primarily as a single band with a molecular weight of 46 kDa. A slight shoulder at ~ 80 kDa can be assigned to a small amount of dimeric protein. 41 ".4 Conclusions We have successfully overexpressed and purified SPL with immobilized metal affinity chromatography. Unlike other published reports12 our SPL was purified with ~3 Fe and 3 82', indicating a significantly higher cluster content than previously reported,and was not subjected to artificial reconstitution. On SDS- PAGE, SPL eluted at 43 kDa, with a purity greater than 95% as analyzed by SDS-PAGE gel electrophoresis. Size exclusion chromatography yields a primarily monomeric protein, with an absorbance at both 280 nm and 426 nm, showing that the iron sulfur cluster is found in the monomeric SPL. A small shoulder is found at ~80 kDa and maybe the result of high protein concentrations used in these experiments. The presence of SPL as a monomeric protein coincides with many other members of the radical SAM superfamily such as PFL-AE13 and MoaA14 and to DNA photolyase, with which SPL shares sequence homology.15 Our SPL proved to be unstable at higher concentrations but the addition of SAM greatly enhanced the stability and solubility and allowed for a 3 fold increase in concentration. The enhanced stability of SPL in the presence of SAM is most likely due to the binding of SAM to SPL, possibly to the iron sulfur cluster. In previous proposals by Mehl and Begley and later by Cheek and Broderick the 7 cluster donates an electron to SAM and causes cleavage.16’ 1 42 ”.5 10. 11. 12. References Voet, D.; Voet, J. G., Biochemistry. 2nd ed.; John Wiley & Sons: New York, 1995. Winstanley, C.; Rapley, R., Protein Derived Cloning Vectors. ln Molecular Biomethads Handbook, Rapley, R.; Walker, J. M., Eds. Humana Press: Totowa, New Jersey, 1998; pp 165-179. Porath, J.; Carlsson, J.; Ollson, l.; Belfrage, G., Metal chelate affinity chromatography, a new approach to protein fractionation. Nature 1975, 258, 598-599. Scopes, R. K., Protein Purification: Principles and Practice. 3rd ed.; Springer: New York, 1994. Miller, J. H., Experiments in Molecular Genetics. C. S. H. Press: New York, 1972. Broderick, J. B.; Henshaw, T. F.; Cheek, J.; Wojtuszewski, K.; Smith, S. R.; Trojan, M. R.; McGhan, R. M.; Kopf, A.; Kibbey, M.; Broderick, W. E., Pyruvate fonnate-lyase-activating enzyme: Strictly anaerobic isolation yields active enzyme containing a [3Fe-48]+ cluster. Biochemical and Biophysical Research Communications 2000, 269, (2), 451-456. Bradford, M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 1976, 72, 248. Fish, W. W., Rapid colorimetric micromethod for the quantitation of complexed iron in biological samples. Methods in Enzymalagy 1988, 158, 357-364. Beinert, H., Methods in Enzymalagy 1978, 54, (435-445). Beinert, H., Semi-micro methods for analysis of labile sulfide and of labile sulfide plus sulfane sulfur in unusually stable iron-sulfur proteins. Analytical Biochemistry 1983, 131, 373-378. Buis, J. M.; Cheek, J.; Kalliri, E.; Broderick, J. B., Characterization fo an active spore photoproduct lyase, a DNA repair enzyme in the radical sam superfamily. Journal of Biological Chemistry 2006, In Press. Rebeil, R.; Nicholson, W. L., The subunit structure and catalytic mechanism of the Bacillus subtilis DNA repair enzyme spore photoproduct lyase. Proceedings of the National Academy of Sciences of the United States of America 2001 , 98, (16), 9038-43. 43 13. 14. 15. 16. 17. Broderick, J. B.; Duderstadt, R. E.; Fernandez, D. C.; Wojtuszewski, K.; Henshaw, T. F.; Johnson, M. K., Pyruvate fonnate-Iyase activating enzyme is an iron-sulfur protein. Journal of the American Chemical Society 1997, 1 19, (31), 7396-7397. Haenzelmann, P.; Schindelin, H., Crystal structure of the S- adenosylmethionine-dependent enzyme MoaA and its implications for molybdenum cofactor deficiency in humans. Proceedings of the National Academy of Sciences of the United States of America 2004, 101 , (35), 12870-12875. Sancar, A., Structure and Function of DNA Photolyase and Cryptochrome Blue-Light Photoreceptars. Chemical Reviews 2003, 103, (6), 2203-2237. Mehl, R. A.; Begley, T. P., Mechanistic Studies on the Repair of a Novel DNA Photolesion: The Spore Photoproduct. Organic Letters 1999, 1, (7), 1 065-1 066. Cheek, J.; Broderick, J. B., Direct H Atom Abstraction from Spore Photoproduct C-6 Initiates DNA Repair in the Reaction Catalyzed by Spore Photoproduct Lyase: Evidence for a Reversibly Generated Adenosyl Radical Intermediate. Journal of the American Chemical Society 2002, 124, (12), 2860-2861. 44 CHAPTER III SOLUBILIZATION OF SPORE PHOTOPRODUCT LYASE ll|.1 Introduction Initial work on the spare photoproduct lyase protein in the Broderick lab yielded a protein that was unstable at room temperature for periods of time greater than 10 minutes and was subject to precipitation at a concentration above ~250 uM (J. Cheek and J. M. Buis unpublished work). This was problematic for a detailed study of the iron sulfur cluster of SPL, as many types of spectroscopy require a high concentration of protein. Protein overexpression and solubility is dependent upon a number of factors, including the E. coli overexpression strain used, the cell lysis procedure, the buffering conditions including pH, salt concentration, glycerol and detergent content, the expression vector in which the gene is inserted, the temperature of cell growth, and the addition of co-factors and co-substrates; all these factors can all have a dramatic effect on overexpression and/or solubility.1 For example, if a protein is in a buffer with a pH close to its isoelectric point it is far less likely to be soluble or stable in solution.2 Besides changing the above conditions, the protein in question can be cc- expressed with other proteins such as iron-sulfur assembly proteins of the isc 45 operon as has been done with BioB.3 In this instance the co-expressed protein aids in cluster assembly, which can be crucial to protein folding in Fe-S proteins. Other ways of increasing solubility include expressing the protein of interest with an additional peptide tag that has shown high solubility. Commercial vectors such as pET44a-c have Nus-tag located in the sequence and when a specific protein is cloned into this expression vector the resulting protein will have this highly soluble tag attached to the c-terminus of the protein, which can greatly enhance the protein solubility. While E. coli are often an ideal expression system for proteins from other organisms, sometimes the protein is unable to overexpress in abundance in E. coli or the protein overexpresses well but is found as inclusion bodies that are located in the cell pellet after lysis.1 There are a few possible explanations for this type of protein behavior. E. coli lacks the tRNA for certain so-called rare codons and is unable to transcribe the mRNA produced by the gene. This has been problematic for certain genes and organisms that are rich is GC sequences or reversely for certain AT-rich genes. Different commercial competent cells have been developed to overcome this obstacle including BL21 CodonPlus cells from Stratagene and RosettaTM cells from Novagen. Finally, the spare photoproduct lyase from B. subtilus has protein homologs among several other sporulating bacteria. It has been found that protein homologs in some species are more soluble in vitro than others.1 46 in the study of spare photoproduct lyase, many of the above schemes have been employed in an attempt to increase protein solubility; most of these approaches were unsuccessful. III.2 Experimental Methods Materials All chemicals were commercially obtained except when noted othenrvise, and were of the highest purity. Expression vectors pET28a, pET44a, pET42a, and pET30 EKILIC were purchased from NovagenTM. E. Cali strains of Novablue, Tuner(DE3)pLysS, Rosetta(DE3)pLysS, and BL21(DE3)CodonPlus were purchased from Novagen. SDS-PAGE (12% Tris-HCI) gels were commercially obtained from BioRad Scientific. FPLC experiments were carried out with an AKTA Basic FPLC from Amersham Biosciences. A BioRad GS-710 Gel imaging densitometer was used to scan SDS-PAGE gels and calculate band densities L ysis procedures Several types of lysis procedures were employed throughout this work and are described below: Lysis procedure 1; whole cell lysis was carried out by removing 1 mL of a cell growth and pelleting at 27,000 g for 5 minutes. 50 uL of a 2X SDS-PAGE dye (100mM Tris-HCI, 4% SDS, 0.4% bromophenol blue, 20% glycerol) was added to cell pellet and cells were resuspended and placed in boiling water for 15 minutes. 47 The sample was centrifuged at 27,000 g for 10 minutes and loaded immediately onto a SDS-PAGE gel. Lysis procedure 2; enzymatic lysis was carried out by pelleting a 50 mL cell culture at 27,000 g for 10 minutes. A lysis buffer containing 3 mL of the following buffer (50 mM Hepes, 250 mM NaCl, 5 mM B-ME,10% glycerol, 1% triton X-100, pH 7.5) was added to the cells in addition to 1 mg of lysozyme, 0.1mg of DNAse I and RNAse A. The cells were mixed at 4 °C for 30 minutes and 10 mM MgCl2 was added followed by an addition 60 minutes of mixing. The cells were centrifuged at 27,000 g for 30 minutes with a 10 uL aliquot taken for SDS-PAGE. Lysis Procedure 3; enzymatic lysis was carried out by pelleting a 50 mL cell culture at 27,000 g for 10 minutes. A lysis buffer containing 3 mL of the following buffer (20 mM sodium phosphate, 500 mM NaCl, 5% glycerol, 1% triton X-100, pH 8.0) was added to the cells in addition to 1 mg of lysozyme, 0.1mg of DNAse I and RNAse A. The cells were mixed at 4 °C for 30 minutes and 10 mM MgCl2 was added followed by an addition 60 minutes of mixing. The cells were centrifuged at 27,000 g for 30 minutes with a 10 pL aliquot taken for SDS-PAGE. Lysis Procedure 4; enzymatic lysis was carried out by pelleting a 1 L cell culture at 27,000 g for 10 minutes. A lysis buffer containing 15 mL of the following buffer (50 mM Hepes, 250 mM NaCl, 10% glycerol, 1% triton X-100, pH 7.5) was added to the cells in addition to 1 mg of lysozyme, 0.1mg of DNAse l and RNAse A. The cells were mixed at 4 °C for 30 minutes and 10 mM MgCI2 48 was added followed by an addition 60 minutes of mixing. The cells were centrifuged at 27,000 g for 30 minutes with a 10 uL aliquot taken for SDS-PAGE. Cloning Bacillus subtilus spIB into pET28a, pE T 42a and pET44a The spIB gene from B. subtilus was acquired by standard polymerase chain reaction (PCR) procedures from the previously cloned expression vector pET14b-spl17 using the DNA primers, 5’-GCCGCGAATTCATGA GAACCCATTTGTl’C-3’ and 5’-GATGCAGAACCCATTTGTTCCGCAGC‘I'I'GT-3' with a EcaR1 and a Hindlll restriction site incorporated into the respective primers. The Hind/II/EcaR1 digested PCR products were cloned into the respective overexpression vectors also digested with HindIII/EcoR1 in pET-42a, pET443, and pET28a using T4 DNA ligase (New England Biolabs) and NovaBlue competent cells. The PCR product was cloned in sequence with pET42a to incorporate a N-terrninal GST tag and a N-tenninal hexahistidine tag, the pET44a was cloned in sequence with a N-terrninal Nus tag and N-terrninal hexahistidine tag, and the pET28a was cloned in sequence with a N-tenninal hexahistidine tag. The resulting constructs (pET-44a/spl1, pET4Za/spl2, pET28a/spl1) were transformed into NovaBlue Escherichia coli for isolation and purification of the plasmid DNA. The constructs were then transformed into Tuner(DE3)pLysS Escherichia coli for protein overexpression. The fidelity of the PCR product was verified by dideoxynucleotide sequencing. 49 Cloning splB from Bacillus halodurans into pET30/EK/LIC Chromosomal DNA from Bacillus halodurans strain (21591 D) was purchased from ATCC and used as the template DNA for standard PCR procedures with the primers 5’GACGACGACAAGATGGAAAATATGTTTAG-3’ and 5’-GAGGAGAAGCCCGG'l'l'TAAA'l'l'ATATAG-3’ which include a specific overhang sequence that takes advantage of the 3’ ——> 5’ exonuclease activity of T4 DNA polymerase. PCR products were treated with the T4 DNA polymerase and annealed to a linear pET30Ek/LIC vector, followed by transformation into NovaBlue competent cells. The spIB gene was cloned in sequence with the pET30EK/LIC with an N-tenninal hexahistidine tag. The constructs were then transformed into Tuner(DE3)pLysS Escherichia call for protein overexpression. The fidelity of the PCR product was verified by dideoxynucleotide sequencing. Transformation of pET1 4B into various competent cell strains The pET14B/SPL17 construct was alternatively transformed into both Rosetta(DE3)pLysS and BL21(DE3)CodonPlus competent cell lines and grown in 1 L of MM media to ODaoo = 0.6 followed by the addition of 0.5 mM IPTG. The culture was grown an additional 3 hours and checked for overexpression on SDS-PAGE gels. Protein dialysis and concentration for varying buffer conditions Spore photoproduct lyase was overexpressed in Tuner(DE3)pLysS E. coli in a 10 L ferrnentor in minimal media as previously noted in Chapter 2 and 50 purified on a Co-TalonTM metal affinity column (BD Biosciences, 10 X 1cm) with a linear gradient between 0% buffer A (50 mM Hepes, 250 mM NaCl, 5 mM [3- ME,10% glycerol, pH 7.5) and 100% buffer B (50 mM Hepes, 250 mM NaCl, 250 mM imidizole,5 mM B-ME,10% glycerol, pH 7.5) run over 60 min after washing with buffer A for 20 min after loading crude lysate and prior to the linear gradient. Fractions with SPL were pooled and concentrated with a YM-10 Amicon concentrator to ~3 mg/mL and dialyzed in Spectra/Par dialysis tubing (molecular weight cutoff 12-14000 kDa, 0.32 mL / cm) with corresponding buffer in 2 X 2 L of buffer for 2 hours. Following dialysis, protein was concentrated with YM-10 Amicon ultra-concentrator. Altered growth conditions of pET14b-spl1 7in Tuner(DE3)pLysS E. coli The spIB gene from Bacillus subtilis used in this work had been previously cloned into the pET14b expression vector from Novagen in our lab by Dr. Jennifer Cheek. A single colony of the overexpression strain pET14b-SPL17 transformed into E. coli Tuner(DE3)pLysS competent cells was used to inoculate 50 mL of Luria/Bertani (Miller’s Modification)4 medium containing 50 ug/mL of ampicillin: Growth condition 1; this culture was grown to saturation at 37 °C and used to inoculate a 10 L flask of minimal media/ampicillin described elsewhere.5 The 10 L culture was grown at 37 °C in a New Brunswick Scientific fennentor (250 rpm, 5 L/min 02). When the culture reached an ODeoo = 0.6, isopropyl B-D- thiagalacto-pyranoside (IPTG) was added to 1 mM final concentration and the 51 medium was supplemented with 750 mg Fe(NH4)2(SO4)2 . The culture was grown for an additional 2 hours, and then was cooled to 25 °C and placed under nitrogen (5 L/min). The culture was further cooled to 15 °C, moved to a 4 °C refrigerator and left under nitrogen overnight for 12 hours. The cells were then harvested by centrifugation at 4°C and stored under nitrogen at —80 °C for further use. Growth condition 2; this culture was grown to saturation and used to inoculate to inoculate a 10 L flask of LurialBertani (Miller’s Modification)4 medium. The 10 L culture was grown at 37 °C in a New Brunswick Scientific ferrnentor (250 rpm, 5 L/min 02). When the culture reached an ODsoo = 0.6, isopropyl B-D—thiogalacto-pyranoside (IPTG) was added to 1 mM final concentration and the medium was supplemented with 750 mg Fe(NH4)2(SO4)2 . The culture was grown for an additional 2 hours, and then was cooled to 25 °C and placed under nitrogen (5 L/min). The culture was further cooled to 15 OC, moved to a 4 °C refrigerator and left under nitrogen overnight for 12 hours. The cells were then harvested by centrifugation at 4°C and stored under nitrogen at - 80 °C for further use. Growth condition 3; this culture was grown to saturation at 37 °C and used to inoculate a 1 L flask of minimal media/ampicillin described elsewhere.5 The 1 L culture was grown at 37 °C in 2.5 L Bellca fermentation flasks in a New Brunswick C24 Incubator Shaker (250 rpm). When the culture reached an ODeoo = 0.6, isopropyl B-D-thiogalacto-pyranoside (IPTG) was added to 1 mM final 52 concentration and the medium was supplemented with 100 mg Fe(NH4)2(SO4)2. The culture was grown for an additional 3 hours. The cells were then harvested by centrifugation at 4 °C and stored under nitrogen at —80 °C for further use. Growth condition 4; this culture was grown to saturation at 37 °C and used to inoculate a 1 L flask of minimal media/ampicillin described elsewhere.5 The 1 L culture was grown at 25 °C in 2.5 L Bellca fermentation flasks in a New Brunswick C24 Incubator Shaker (250 rpm). When the culture reached an ODsoo = 0.6, isopropyl B-D-thiogalacto-pyranoside (IPTG) was added to 1 mM final concentration and the medium was supplemented with 100 mg Fe(NH4)2(SO4)2. The culture was grown for an additional 5 hours. The cells were then harvested by centrifugation at 4 °C and stored under nitrogen at —80 °C for further use. Growth condition 5; this culture was grown to saturation at 37 °C and used to inoculate a 1 L flask of minimal media/ampicillin described elsewhere.5 The 1 L culture was grown at 16 °C in 2.5 L Bellca fermentation flasks in a New Brunswick C24 Incubator Shaker (250 rpm). When the culture reached an ODsoo = 0.6, isopropyl B-D-thiogalacto-pyranoside (IPTG) was added to 1 mM final concentration and the medium was supplemented with 100 mg Fe(NH4)2(SO4)2. The culture was grown for an additional 12 hours. The cells were then harvested by centrifugation at 4°C and stored under nitrogen at —80 °C for further use. Growth condition 6; this culture was grown to saturation at 37 °C and used to inoculate a 10 L flask 2 X YT medium. The 10 L culture was grown at 37 °C in a New Brunswick Scientific ferrnentor (250 rpm, 5 L/min 02). When the culture 53 reached an ODsoo = 0.6, isopropyl B-D-thiogalacto-pyranoside (IPTG) was added to 1 mM final concentration and the medium was supplemented with 750 mg Fe(NH4)2(SO4)2 . The culture was grown for an additional 2 hours, and then was cooled to 25 °C and placed under nitrogen (5 lein). The culture was further cooled to 15 °C, moved to a 4 °C refrigerator and left under nitrogen overnight for 12 hours. The cells were then harvested by centrifugation at 4°C and stored under nitrogen at -80 °C for further use. Protein and iron assays Routine determinations of protein concentrations were done by the method of Bradford, using a kit sold by Bio-Rad and bovine serum albumin as a standard.6 Iron assays were performed using the methods of Fish or Beinert.7' 8 Sulfide assays were carried out as previously described by Broderick et al.5 using a modification of the method developed by Beinert.9 Ill.3 Results and Discussion Cloning of Bacillus subtilus spIB into pETZBa, pET4Za, and pET44a The splB gene was successfully cloned into the pET28a, pET4Za, and pET44a expression vectors as monitored by dideoxynucleotide sequencing. The plasmids pET28a-spl1 and pET4Za-spl1 were transformed into Tuner(DE3)pLysS E. coli and grown as described in experimental methods section (growth condition 3). The cells were then lysed with lysis procedure 1 and 54 Figure lll.1 SDS-PAGE of SPL from B. subtilus cloned into pET44a, pET42a, pET28a. A. The overexpression of pET44a-spl1, lanes 1 and 2 are uninduced cell cultures. Lanes 3 and 4 show induced cell cultures (whole cell lysis, procedure 1) to contain a large band at ~66 kDa. Lanes 5 and 6 are induced cell cultures enzymatically Iysed (lysis procedure 4). Lane 7 is the BioRad broad range protein standard. Lanes 8 and 9 are cells enzymatically Iysed (lysis procedure 2) B. The growth and whole cell lysis of pET28a-spl1 and pET42a-spl1 shows no significant overexpression of any band at the expected molecular weights of ~43 kDa and 55 kDa respectively; Lanes 1 is uninduced pET28a-spl1 cells and lane 2 is induced cells (growth condition 3, lysis procedure 1). Lanes 1 is uninduced pET42a-spl1 cells and lane 2 is induced cells (growth condition 3, lysis procedure 1). 55 1 2 3 4 5 6 7 8 9 1O 45kDa Figure "L1 56 loaded and run on 12% Tris-HCI SDS-PAGE gels. The SDS-PAGE gels show no expression of spare photoproduct lyase (Figure llI.1.B). The plasmid pET44a-spl1 was transformed into Tuner(DE3)pLysS E. coli and grown as described in experimental methods section (growth condition 3). The cells were then lysed with lysis procedure 1 and 4 and loaded and run on 12% Tris-HCI SDS-PAGE gels to check for overexpression and solubility of SPL (Figure llI.1.A). As the pET44a plasmid encodes for the addition of a 54 kDa protein sequence in addition to the 42 kDa sequence of SPL, we expect a protein of ~96 kDa. However, SDS-PAGE gel with lysis procedure 1 shows a protein at ~66 kDA, which may be caused by improper transcription of the plasmid. After cell lysis with procedure 4, the 66 kDa band disappears and appears insoluble. These results are summarized in Table "L1 and shows the amount of SPL overexpressed and solubilized as a percentage of the total protein present. Cloning spIB from Bacillus halodurans into pET30/EK/LIC The spIB gene of Bacillus halodurans was successfully incorporated into the overexpression vector pET30/Ek/LIC as monitored by dideoxynucleotide sequencing. This plasmid was transformed into Tuner(DE3)pLysS and grown and overexpressed using growth condition 3. The SDS-PAGE gel of the whole cell lysis (lysis procedure 1) shows a strongly expressed band at ~43kDa as shown in Figure III.2. However, enzymatic cell lysis (lysis procedure 4) yielded no soluble protein as monitored by SDS-PAGE. Further growths (growth conditions 4 and 5) were carried out at lower temperatures at 16 °C and 25 °C to help 57 prevent inclusion body formation but results did not improve. Overall, while the protein was strongly overexpressed, there was no soluble protein or reddish- brown color that would indicate the presence of an abundant iron sulfur cluster containing protein. Organism Expression Vector E. coli Strain Overexpression: SPL as an approximate % of total cell protein Solubility after lysis: SPL as an approximate % of total soluble protein Bacillus subtilis pET 14B Tuner(DE3)pLysS 5% 5% Bacillus subtilis pET 14B Rosetta(DE3)pLyS 4% 4% Bacillus subtilis pET 14B BL21(DE3)CodonPlus 4% 4% Bacillus subtilis pET 14B BL21(DE3)pLysS 3% 3% BaciI/lus halodurans pET 30EK/LIC Tuner(DE3)pLysS 35%: 0% Bacillus subtilis pET 28a Tuner(DE3)pLysS 0% 0% Bacillus subtilis pET42a Tuner(DE3)pLysS 0% 0% Bacillus subtilus pET44a Tuner(DE3)pLysS 30% 0% Table II|.1 vectors, competent cells and organisms. 58 Overexpression and solubility results from the various expression Figure III.2 SDS-PAGE of SPL from B. halodurans overexpressed and Iysed. A. The addition of IPTG, lanes 1, 3, and 5, causes significant overexpression versus cultures (lanes 2, 4, 6) without IPTG (lysis procedure 1). Lane 7 is the BioRad protein standard. B. After cell lysis (procedure 4) no overexpressed protein is found in the supernatant, lanes 3 and 4. Most of the protein is found in the cell pellet, lanes 1 and 2. Lane 5 is the BioRad protein standard. 59 45kDa 45kDa qumdm3 60 Overexpression of spIB in Rosetta and CodonPlus competent cells The pET14/SPL17 construct was successfully transformed into both Rosetta(DE3)pLysS and BL21(DE3)CodonPlus compentent cells. The resulting colonies were checked for SPL overexpression in LB media via growth condition 4 and subsequent lysis by method procedure 1 as described in the experimental methods. For both the Rosetta(DE3)pLysS and BL21(DE3)CodonPlus compentent cells, there is little overexpression (Figure Ill.3) of the 42 kDa SPL protein and it is not an improvement upon the Tuner(DE3)pLysS cells that had been used prior to this, suggesting that rare codons are not the reason for SPL's lack of expression. These results are summarized in Table "L1 45 kDa Figure Ill.3 SDS-PAGE gel of pET14b-spl17 transformed and overexpressed in Rosetta and CodonPlus competent cells. Lane 1 and 2, induced and uninduced SPL in Rosetta competent cells. Lane 3 and 4, induced and uninduced SPL in Codon Plus competent cells. Lane 5, BioRad broad range protein Standard. 61 Differing buffer conditions of the pET14b/spl17 construct Varying the buffer conditions and their affect on the solubility of the spare photoproduct lyase protein are summarized in Table III.2. The protein was initially all purified in the same manner with the same buffer and then dialysis was used to change the buffer conditions followed by protein concentration. Most of the buffer modifications did not yield improvement in protein solubility while other significantly hindered the stability of the protein. However, the addition of SAM to SPL greatly enhanced the protein stability and allowed for concentrations upwards of 750pM. Buffer Change [SPL] Max 0 mM imidizole 185 uM 0 mM imidizole, 0 mM B-ME 183 uM 50 mM Tris 103 uM 30% glycerol 125 pM 20% glycerol 250 (M 5% glycerol 46 uM pH 7.0 low 200 mM NaCl 108 pM 100 mM NaCl 97 uM 1%CHAPS as detergent low 1%Triton X-100 as detergent 100 uM 3mM SAM 750 pM 1mM EDTA 150 pM 5% Ethanol low Table III.2 The variance of buffering conditions and its affect on SPL. Buffer: 50mM Hepes, 300mM NaCI, 5mM B-ME, 10% glycerol, pH 7.5, ~125mM imidizole, gives a best concentration of 150 pM before precipitation. The increase in glycerol concentration was one of the few effective ways in raising the solubility of SPL. However, addition of SAM was most effective at stabilizing SPL. 62 Effect of altered growth conditions an overexpression of pET14b-spl1 7 Several proteins have been shown to have better overexpression when expressed in different medium or at different growth temperatures. Given this consideration, we have grown the pET14b-spl17 in the E. coli strain Tuner(DE3)pLysS and varied both the temperature and the growth media. These results are summarized in Table Ill.3 with growth conditions corresponding to those described in the experimental methods. Growth Conditons Amount of SPL as a % of total cell protein SPL Growth 1 37 °C, MM media, 2 Hr induction O/N anaerobic incubation 10 L Ferrnentor 5% SPL Growth 2 37 °C, LB Media, 2 Hr induction OIN anaerobic incubation 10 L Ferrnentor 2% SPL Growth 3 25 °C, MM media, 5 Hr induction 1 L Bellca Flask 5% SPL Growth 4 16 °C, MM media, 12 Hr induction 1L Bellca Flask 4% SPL Growth 5 37 °C, MM media, 2 Hr induction O/N anaerobic incubation 10 L Fennentor 2% Table Ill.3 Amount of SPL present in whole cell lysis on SDS-PAGE gels as a percentage of the total cellular protein. 63 lll.4 Conclusions The cloning of spIB from Bacillus subtilis into the different expression vectors pET 44a, pET 423 and pET 28a did not yield any soluble protein that was produced in sufficient quantity for purification. Whole cell SDS-PAGE lysis of the pET44a construct yield a substantial amount of protein but none of this proved to be soluble and was primarily found in the cell pellet after lysis and centrifugation. Whole cell SDS-PAGE of the pET 42a and pET28a constructs indicated no protein overexpression at the expected molecular mass. The cloning of the spIB gene from Bacillus halodurans into the expression vector pET30 EKILIC was successful but also did not yield any soluble protein. Attempts at altering growth conditions did not improve protein overexpression. Cells grown with the constructs and the whole cell lysis wear not of a dark brownish red color that would indicate an abundance of protein with an iron sulfur cluster present. The use of the different E. coli competent cell strains Rossetta(DE3)pLysS and BL21(DE3)CodonPlus did not have much of an affect on protein overexpression and were not an improvement over the previously used Tuner(DE3)pLysS cells. Varying the buffer conditions of purified SPL from Bacillus subtilis did not yield substantial improvement in the solubility of SPL with the exception of addition of SAM which yielded a 3 fold increase in solubility to ~750 uM. This is 64 a notable result as SAM is the expected cofactor for SPL and may indicate that the presence of SAM in some way can stabilize SPL. 65 llI.5 References Scopes, R. K., Protein Purification: Principles and Practice. 3rd ed.; Springer: New York, 1994. Voet, D.; Voet, J. G., Biochemistry. 2nd ed.; John Wiley & Sons: New York, 1995. Casper, M. M.; Jameson, G. N. L.; Davydov, R.; Eidsness, M. K.; Hoffman, B. M.; Huynh, B. H.; Johnson, M. K., The [4Fe-4S]2+ Cluster in Reconstituted Biotin Synthase Binds S-AdenosyI-L-methionine. Journal of the American Chemical Society 2002, 124, (47), 14006-14007. Miller, J. H., Experiments in Molecular Genetics. C. S. H. Press: New York, 1972. Broderick, J. B.; Henshaw, T. F.; Cheek, J.; Wojtuszewski, K.; Smith, S. R.; Trojan, M. R.; McGhan, R. M.; Kopf, A.; Kibbey, M.; Broderick, W. E., Pyruvate forrnate-lyase-activating enzyme: Strictly anaerobic isolation yields active enzyme containing a [3Fe-4$]+ cluster. Biochemical and Biophysical Research Communications 2000, 269, (2), 451-456. Bradford, M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 1976, 72, 248. Beinert, H., Methods in Enzymalagy 1978, 54, (435-445). Fish, W. W., Rapid colorimetric micromethod for the quantitation of complexed iron in biological samples. Methods in Enzymalagy 1988, 158, 357—364. Beinert, H., Semi-micro methods for analysis of labile sulfide and of labile sulfide plus sulfane sulfur in unusually stable iron-sulfur proteins. Analytical Biochemistry 1983, 131, 373-378. 66 CHAPTER IV SPECTROSCOPIC CHARACTERIZATION OF THE IRON SULFUR CLUSTER IN SPORE PHOTOPRODUCT LYASE |V.1 Introduction The iron sulfur clusters of proteins have been characterized by a variety of spectroscopic techniques including ultra-violet/visible spectroscopy, electron paramagnetic resonance and Méssbauer spectroscopy." 2 3 The UV/visible absorption spectroscopy of iron sulfur cluster has been well characterized over the years and is dominated by ligand to metal charge transfer (LMCT) bands between the sulfurs and the iron. The number of peaks generated by this LMCT causes the spectra to have broad absorption from 300 nm to 600 nm. Specific features are dependent upon the type and oxidation state of the cluster. It is, however, still difficult to determine precisely what type of iron sulfur cluster is present and what kind of ligands might be bound to the cluster due to the abundance of transitions present. Although UV/visible spectroscopy can provide insight as to whether a cluster is present and is redox active other techniques are needed to fully understand the electronic structure of an iron sulfur cluster.2 Electron paramagnetic resonance (EPR) spectroscopy is a more specific technique that probes the interaction between paramagnetic centers and an 67 applied magnetic field. It is applicable to any species containing one or more unpaired electrons, whether this is in the form of an organic radical or the Protein Cluster Oxidation Formal EPR Massbauer State Valence g values Isomer shift (temp) (mm/sag Rubredoxin 1Fe-OS Oxidized 1Fe3+ 4.3, 9 0.25 (< 20 K) Reduced 1 Fe2+ None 0.65 2-Iron 2Fe-28 Oxidized 2Fe3+ None 0.26 ferredoxin Reduced 1Fe3+ 1.89, 1.95, 2.05 0.25, 0.55 1F 62+, (<100 K) 3-Iron 3Fe-4S Oxidized 3Fe3+ 1.97, 2.00, 2.02 0.27 ferredoxin (<20 K) Reduced 2Fe3+ None 0.30, 0.46 1 Fe2+ 4-lron 4Fe-48 Oxidized 3Fe3fi 2.04, 2.04, 2.12 0.31 ferridoxln 1Fe2+ (<100 K) lntermed. 2Fe3+ None 0.42 2Fe2+ Reduced 3+ 1.88, 1.92, 2.06 0.57 1Fe , 3Fe2+ Table IV.1 Typical spectroscopic properties of iron sulfur clusters.4 unpaired electrons of a transition metal center. An electron has a magnetic moment. When placed in an external magnetic field of strength Bo, this magnetic moment can align itself parallel or antiparallel to the external field. The former is a lower energy state than the latter (this is the Zeeman effect), and the energy 68 separation between the two is AE = 9641380, where g, is the gyromagnetic ratio of the electron, the ratio of its magnetic dipole moment to its angular momentum, and [13 is the Bohr magneton. To move between the two energy levels, the electron can absorb electromagnetic radiation of the correct energy: AE = hv = gepro (1) and this is the fundamental equation of EPR spectroscopy. The paramagnetic centre is placed in a magnetic field and the electron caused to resonate between the two states; the energy absorbed as it does so is monitored, and converted into the EPR spectrum. When an unpaired electron is in an atom, it feels not only the external magnetic field Bo applied by the spectrometer, but also the effects of any local magnetic fields. Therefore, the effective field Ben felt by the electron is: Beff = Bo(1 - 0) (2) where 0 allows for the effects of the local fields (it can be positive or negative), and therefore the resonance condition is A5 = hv = QePBBeff = 9903800 - 0) (3) The quantity ge(1 - a) is called the 9 factor, given the symbol 9, so A5 = “V = til/830 (4) Given this last equation, you can measure g from the EPR experiment by measuring the field Bo and the frequency v at which resonance occurs. If g differs from g.3 (2.0023), this implies that the ratio of the electron's magnetic moment to 69 its angular momentum has changed from the free electron value. Since the electron's magnetic moment is constant (it's the Bohr magneton), then the electron must have gained or lost angular momentum. It does this through spin- orbit coupling, and because the mechanisms of spin-orbit coupling are well understood, the magnitude of the change can be used to give information about the nature of the atomic or molecular orbital containing the electron." 2 EPR has proven to be a highly useful technique for examining iron sulfur cluster containing proteins. Although the spin states of the iron found in the cluster is typically S = 2 or S = 5/2 for the Fe2+ and Fe3+ oxidation states, the Fe exhibits antiferromagnetic coupling between the different irons present. Thus, the spin for a typical [4Fe-4S]1+ cluster is S =1/2 while the spin of a [4Fe-S]2+ cluster is S = 0 (EPR silent). As such, some types of cluster are not observable by EPR spectroscopy. Despite this limitation, EPR provides powerful insight concerning the type of cluster, oxidation state of the cluster, and the cluster’s surrounding environment.1' 4 Table IV.1 illustrates the different 9 values obtained of proteins containing different types of iron sulfur clusters.4 In order to get a more complete view of the cluster content of a protein such as SPL, an additional spectroscopic technique like MOssbauer spectroscopy can be utilized to complement the EPR. Méssbauer spectroscopy is one of the most useful tools in the study of iron sulfur cluster and is capable of detecting any iron center in a protein regardless of the oxidation state or magnetic properties of the iron. Thus, states such as the [4Fe-4S]2+ cluster can be detected." 2 Méissbauer spectroscopy takes advantage of the recoilless 7O nuclear gamma resonance and in isotopes with a nuclear spin, such as 57Fe, it can be used to detect transitions between the nuclear ground state and nuclear excited state which is 14.4 KeV for 57Fe. The Miissbauer phenomenon rests on the fact that in a solid most of the recoil energy is converted into lattice vibrational energy. In a bare nucleus, one would observe only a single transition, however, when embedded in a environment with symmetry lower than spherical, tetrahedral or cubic, the degeneracy of the nuclear excited state is lifted by the quadrupole moment interaction with the electric field gradient, providing the characteristic doublet associated with a MOssbauer spectrum. The splitting of the signal can be measured and is known as AEQ. Another useful measurement is the isomer shift (6) which arises from the differences in s-electron density at the nucleus created by varying environments. However, because the radial distribution of the d and s orbitals overlap, 8 is also a good indicator of the oxidation state of the cluster.5 Table IV.1 shows some typical values for the isomer shifts of iron sulfur clusters in proteins. Higher isomer shifts are indicative of a more electron rich cluster environment. Taken together, the isomer shift and AEQ can give indications to the oxidation state, spin state, degree of covalency and coordination environment. The information from M6ssbauer spectroscopy can be enhanced by the magnetic hyperfine splitting that results from the presence of an unpaired electron close to the nucleus and in combination with EPR can help deduce the type of cluster present." 5 71 The combination of the methods above, have been employed by our lab to study both the as-isolated SPL and chemically reduced SPL. Previous studies have shown that SPL contains a redox active iron sulfur cluster that can undergo cluster transformation.6 However, due to the difficulty of overexpressing and purifying SPL, these studies were carried out with either inactive protein or artificially reconstituted protein.6 These studies also did not effectively account for what percentage of the enzyme’s cluster was in which state. Our work herein was designed to sort out which type of clusters are present in both as purified SPL and in active SPL under reducing conditions. The protein has also been examined in the presence of the SPL’s cofactor SAM. IV.2 Experimental Methods Materials All chemicals used were commercially purchased and of the highest purity except where otherwise noted. 57Fe was purchased from Cambridge Isotope Laboratories. S-adenosylmethionine was enzymatically synthesized as described below. Synthesis of S—adenasyI-L-methionine SAM was synthesized enzymatically by using the following procedure. A 10mL reaction of 100 mM Tris HCI (pH 8.0) containing 50 mM KCI, 26 mM MgCl2, 13.0 mM adenosine triphosphate, 8% B-mercaptoethanal, 1mM EDTA, 10.0 mM methionine, 2.5 pL inorganic pyrophosphatase (0.25 U) and 1 mL SAM 72 synthetase crude Iysate were stirred at room temperature for 16 hrs and quenched with 1 mL 1M HCI. The reaction was monitored by thin layer chromatography to completion and the SAM was purified by loading onto a Source 158 cationic exchange column (Pharrnacia, 1 cm x 10 cm) charged with 1M HCI and equilibrated with M0 water. A linear gradient of MO H20 to 1 M HCI was used to elute the SAM. The fractions containing SAM were lyophilized and redissolved in 50 mM HEPES, 200 mM NaCl (pH 8.0). Growth and purification of 57Fe spore photoproduct lyase Growth and purification of 57Fe SPL was carried out as previously stated in Chapter ".2 with the modification of removing all iron from the minimal media and supplementing with 57Fe. The 57Fe stock solution was made by dissolving 57Fe metal in 3:1 MQ H20 to H2804. The final concentration of 57Fe in the growth media was 20 uM. U V/Visible spectroscopy UV-Visible spectra were recorded on a HP-8453 diode-array spectrophotometer. All samples were prepared anaerobically in a mBraun box and transferred to the spectrophotometer with a rubber septum on top of the cuvette. A 1 mL sample of SPL was prepared at a concentration of 65 uM and measured in a quartz cuvette with a 1 cm path length. The SPL sample was titrated with increasing amounts of sodium dithionite and DTT and checked for cluster reduction. 73 Electron paramagnetic spectroscopy EPR measurements were obtained at X-band on a Bruker ESP300E spectrometer equipped with a liquid He cryostat and a temperature controller from Oxford Instruments. Spectra were recorded at 12 K for the [4Fe-4S]1+ and the [3Fe-4S]1+ clusters. The double integrals of the EPR signals were evaluated by using a computer on-Iine with the spectrometer. Spin concentration in the protein samples was determined by calibrating double integrals of the EPR recorded under non-saturating conditions with a standard sample of 0.1 mM Cu (II) and 1 mM EDTA. EPR samples were prepared using 0.35 mM SPL. Samples were reduced with 10 mM sodium dithionite and 10 mM DTT. SAM (3mM) was added to some samples. All samples contained 300 uL of protein solution and were transferred to a 4 mm quartz EPR tube (Wilmad). Ma'ssbauer spectroscopy Samples for Miissbauer spectroscopy were prepared with 57Fe SPL at a concentration of ~0.7 mM in 500 uL Delran cups with 450 pL of protein solution. Reduced samples were made by adding 10 mM sodium dithionite and 10 mM DTT and incubating for 10 minutes on ice. SAM (3 mM) was added to some samples. Méssbauer spectra were recorded at Emory University in either a weak- field spectrometer equipped with a Janis 8DT variable-temperature cryostat or a strong-field spectrometer furnished with a Janis CNDT/SC SuperVaritemp 74 cryostat encasing an 8-T superconducting magnet. Both spectrometers operate in a constant acceleration mode in a transmission geometry. Méssbauer data analysis was performed by Ricardo Garcia and Vincent Huynh at Emory University. IV.3 Results and Discussion Synthesis of S-adenasyl-L-methianine The SAM synthesis reaction and purification (Scheme IV.1) is currently carried out as described in the Experimental Methods. SAM elutes as a broad band from the cation exchange column between 0.4 and 0.6 M HCI (Figure IV.1). The isolated SAM was lyophilized to yield a crystalline colorless solid, presumably in the chloride salt form. A total of 35 mg was obtained corresponding to 70% reaction yield. SAM Synthetase Methionine + ATP -OCC NH3+ Scheme IV.1 Generalized reaction scheme for the enzymatic synthesis of AdoMet. 75 Au 0 3O 6O 90 1 20 min Figure IV.1 Purification of SAM by cation exchange chromatography. SAM elutes as a large broad peak (A260) between approximately 0.4 and 0.6 M HCI on a source 15s cationic exchange column with a linear gradient between MQ H20 and 1M HCI. U V/visible spectroscopy The UV-visible spectrum of the purified enzyme (Figure IV.2) is characteristic of the presence of an iron-sulfur cluster, although it is not particularly definitive of a specific cluster type. The spectrum exhibits a broad shoulder with maxima at 410 nm (11.9 mM’1cm'1) and 450 nm (10.5 mM'1cm'1), similar to what has been observed for anaerobically purified pyruvate forrnate- lyase activating enzyme7 and lipoyl synthase.8 This signal can be reduced by the addition of dithionite as seen in Figure IV.2 with the loss of the shoulder peaks 76 typical of a reduction to a [4Fe-4S]1+ cluster. The UVNis thus confirms the presence of an iron sulfur cluster and that the iron sulfur cluster is redox active. 300 400 500 600 700 800 Figure IV.2 UV-visible absorption spectra of SPL as isolated (solid line) and reduced with dithionite (dashed line). For both spectra, the protein was 65 pM in 20 mM sodium phosphate/500 mM NaCl/5 mM dithiothreitol/5% glycerol, pH 8.0. The reduced protein also contained 5 mM dithionite. The spectra were recorded in a 1 cm pathlength cuvette under anaerobic conditions at room temperature.9 Electron paramagnetic spectrascap y SP lyase exhibits a strong, nearly isotropic electron paramagnetic resonance (EPR) signal which is centered at g = 2.02 and observable only below 35 K (Figure IV.3). The 9 value, the low anisotropy, and the temperature dependence are consistent with the assignment to a [3Fe-4S]1+ cluster being 77 present in the as isolated form of the enzyme. Spin quantification of the [3Fe- 4S]1+ show that it accounts for up to 35% of the total iron. SP lyase can be reduced under anaerobic conditions by titration with sodium dithionite (Figure IV.2). The work presented in this section on the UVNisibIe spectroscopy of SPL was published in the Journal of Biological Chemistry.9 a '75 C .9. C E J LIJ 2800 3200 3600 4000 Field (gauss) Figure IV.3 X-band EPR spectrum of anaerobically isolated SPL. The protein was 350 uM in 20 mM sodium phosphate/500 mM NaCI/10 mM dithiothreitol/5% glycerol, pH 8.0. Conditions of measurement, T=12 K microwave power, 2 mW; microwave frequency, 9.4841 GHz; modulation amplitude, 10.084; and receiver gain, 2 x 104, 1 scan accumulated.9 This reduction results in a dramatic change in the EPR spectral properties (Figure IV.4). Rather than the fairly intense, nearly isotropic signal observed for the as-isolated enzyme, the reduced enzyme has a weaker, nearly axial signal 78 that is characteristic of a [4Fe-4S]1+ cluster, with gz = 2.025, gy = 1.928, and 9x = 1.890 (based on simulations, data not shown). The relaxation properties of this signal are also consistent with its assignment as a [4Fe-4S]1+ cluster, as the signal broadens above 20 K and is unobservable above 40 K. Spin quantification of the [4Fe-4S]1+ cluster shows that it accounts for up to 54% of the total iron present in the protein Dithionite reduction of SP lyase in the presence of its cofactor S-adenosylmethionine results in a [4Fe-4S]1+ EPR signal essentially EPR Intensity 2800 3200 3600 4000 Field (gauss) Figure IV.4 X-band EPR spectrum of reduced SP lyase with and without AdoMet. The protein was 350 uM in 20 mM sodium phosphate/500 mM NaCl/10 mM dithiothreitol/5% glycerol, pH 8.0. Dithionite was added to 5 mM (both spectra) and AdoMet to 2 mM (lower spectrum only). Conditions of measurement, T = 12 K microwave power, 2 mW; microwave frequency, 9.4841 GHz; modulation amplitude, 10.084; and receiver gain, 2 x 104, 1 scan accumulated. 79 identical to that seen in the absence of SAM, albeit with a lower signal intensity. The EPR spectra of SPL are similar to those observed in previous studies of reconstituted SPL,6 with a [3Fe-4S]1+ cluster being observed prior to reduction. After reduction, the [3Fe-4S]1+ cluster is transformed to a [4Fe-4$]1+. The [3Fe- 4S]1+ however only accounts for one third of the total iron in the as-isolated sample, with the remaining being present in an EPR silent form. The EPR silent iron is also likely in an iron sulfur cluster as the Fe:S ratio is 1:1. SPL gains both catalytic activity (Chapter V) and a [4Fe-4S]1+ cluster upon reduction, consistent with previous work demonstrating the presence of a catalytically relevant [4Fe- 4S]1+ cluster in the radical SAM enzymes PFL-AE1o and LAM.11 Our data suggests that this [4Fe-4S]1+ cluster is generated by reductive cluster conversion of our [3Fe-4S]1+ cluster because of the loss of this signal after reduction and the appearance of a [4Fe-4S]1+ cluster signal. In contrast to other members of the radical SAM superfamily, addition of SAM to the reduced SPL does not alter the line shape or g values of the [4Fe- 4S]1+ EPR signal; however, it does result in a decrease in the intensity of the signal (Figure NA). This reduction in intensity in the presence of SAM has previously been observed for SPL;6 one interpretation of this observation could be that there is nonproductive reductive cleavage of SAM (and corresponding oxidation of the cluster). We have, however found no evidence for SAM cleavage in the presence of SPL (Chapter VI), indicating that other explanations such as a change in spin state of same population of the cluster, must be considered. Both 80 Q-band EPR (Chapter VIII) and MOssbauer spectroscopy provide some early evidence for this possibility. The work presented in this section on the EPR spectroscopy of SPL was published in the Journal of Biological Chemistry.9 Mc'issbauer spectrascap y M6ssbauer spectroscopy of the as-isolated SPL demonstrates the presence of multiple types of iron sulfur cluster (Figure IV.5.A). Simulations of the iron sulfur content show it to contain 53% [2Fe-ZS]2+ cluster with 6 = 0.28 mm/s; AEQ = 0.62mm/s; F = 0.43mm/s. The rest of the iron is a paramagnetic species as noted by the splitting of the quadrupole doublet; this is most likely a [3Fe-4S]1+ cluster, with maybe some admixture of [4Fe-4$]1+. MOssbauer spectroscopy of chemically reduced SPL also demonstrated a mixture of cluster states (Figure IV.5.B). This spectrum can be simulated and contains 10% [4Fe-4S]2+ cluster with 6=0.49mm/s, AEQ=1.11mmIs, and F=0.43mm/s. The other 90% of the iron is a [4Fe-4S]1+ cluster. The spectrum is simulated using two equal intensity S = 1/2 centers, with 6 = 0.60 mm/s, AEQ = 1.29 mm/s, and A = (-30, -22, -11)kG, and 6 = 0.55mm/s AEQ=0.66mm/s A=(2, 15, 18)kG. Note that the isomer shift (6) of both clusters are unusually high, which is indicative of an electron-rich environment. As shown in Table IV.1, typically for a [4Fe-4S]2+ cluster, 6 = 0.42 and 6 = 0.57 for a [4Fe-4S]1+ 81 \\ l \ l 1 Absorption (%) Velocity (mm/s) Figure IV.5 Méssbauer spectra of SPL. A. Native state SPL. The dashed black line is the actual experimental spectrum. The smoothed line is a simulated spectrum of a [4Fe-4S]2+ cluster, weighted to 53% of the iron and the black line is the subtraction of the dashed black line and the simulation. The remaining is indicative of a [3Fe-4S]1+ cluster. B. Reduced SPL. The dashed black line is the experimental spectrum, overlaid on this spectra (black line) is the addition of two simulated clusters. the upper line being a [4Fe-4S] + cluster and the lower being a [4Fe-4S]1+ cluster weighted to 10% and 90% of the total iron. C. Reduced SPL with SAM (dashed black line) and a simulation for a [4Fe-4S]2+ cluster (smoothed line) weighted to 33% of the iron. The black line is the subtraction of the two above spectra, and is indicative of a [4Fe-4S]1+ cluster. Mbssbauer spectroscopy of the chemically reduced SPL with SAM, as with the above spectra, shows a mixture of cluster states (Figure IV.5.C). 82 Simulations of the spectra Indicate that 33% of the iron is in a [4Fe4S]2+ cluster, with the same parameters as in B, 6 = 0.49mm/s, AEQ = 1.11mm/s, and F = 0.43mm/s. The rest is most likely a [4Fe-4S]1+ cluster, the spectnrm of which is different from the ones in Figure IV.5.A and B. This may indicate that the SAM is bound to the cluster. If the bound state is confirmed, the question remains as to why some [4Fe-4S]1+ clusters stay bound to SAM while others are oxidized to the [4Fe-4S]2+ state. IV.4 Conclusions Through the use of several spectroscopic techniques it has been demonstrated that our purified SPL contains an iron sulfur cluster without having to artificially reconstitute the site as has been previously described.6 UV/visible spectroscopy indicates the presence of an iron sulfur cluster with a broad absorption band between 300 nm and 600 nm but does not give any indication as to the type of cluster present. As with other members of the radical SAM superfamily and with previous work on SPL6, the cluster can be reduced, showing it to be a redox active iron sulfur cluster.12 EPR of the as-isolated SPL yielded a spectrum consistent with that of a typical [3Fe-4S]1+ cluster. However, the spin quantification showed that it accounted for only ~25-35% of the total iron present in the protein, meaning that the rest of the iron must be present in some sort of EPR silent state. EPR of the reduced cluster yields a spectrum characteristic of a [4Fe-4S]1+ cluster but again 83 only up to 54% of the cluster is present in this state indicating the presence of another form of cluster. M6ssbauer spectroscopy of SPL grown in 57Fe enriched media gives further insight into the cluster state of the SPL. This indicates that the native state protein is a mixture of a [2Fe-ZS]2+ cluster and a [3Fe-4S]1+ cluster. This is somewhat in line with the EPR data that indicates a [3Fe-4S]1+ and a EPR silent state. Upon reduction the majority of the cluster is changed to a [4Fe-4S]1+ cluster with a high isomer shift. A summary of the different cluster states observed by both EPR and Méssbauer spectroscopy is provided in Table IV.2. As-isolated SPL Reduced SPL Reduced SPL + SAM EPR 35% [are-43]1+ 54% [4r=e-4si1+ 11% [4i=e-4si1+ 65% EPR silent 46% EPR silent 89% EPR silent M6ssbauer 53% [2i=e-2si1+ 10% [4Fe-4S]2+ 33% [4Fe-4siz“ 47% [3Fe-4S]1+ 90% [4i=e-4S]1+ 67% MFG-4511+ Table IV.2 Percentage of total iron present in the iron sulfur cluster of SPL as monitored by EPR and Méssbauer spectroscopy. The high isomer shift is indicative of an electron rich cluster environment which may facilitate the transfer of an electron from the cluster of SPL to SAM and cause cleavage to methionine and the putative 5’-deoxyadenosyl radical during enzymatic turnover. 84 The facile cluster rearrangement that is observed in SPL by both EPR and MOssbauer spectroscopies has been seen in other radical SAM superfamily members including PFL-AE7 and BioB13 and may be a characteristic of the superfamily as a whole. This type of cluster conversion has also been seen in the well studied aconitase enzyme.14 The presence of multiple cluster states is likely the result of cluster oxidation during purification and growth because of the non- cysteinal iron site that is found in the superfamily. Finally the spectrum of reduced SPL and SAM indicates same cluster oxidation to a [4Fe-4S]2+ cluster as well as an iron sulfur cluster with different parameters from the spectrum of the reduced SPL. This is likely the result of a interaction between the cluster and SAM, although it is still unknown as to what type of interaction is occurring. Another possibility is that the presence of AdoMet may cause same population of the cluster to change to a higher spin state, which would be consistent with the reduction in signal observed by EPR of [4Fe-4S]1+ cluster in the presence of SAM. 85 |V.5 10. 11. References Que, L., Physical Methods in Biainarganic Chemistry. University Science Books: Sausilito, CA, 2000. Solomon, E. l.; Lever, A. B. P., Inorganic Electronic Structure and Spectroscopy. Wiley-lnterscience: New York, 1999; Vol. I and II. Rao, P. V.; Holm, R. H., Synthetic Analogues of the Active Sites of Iron- Sulfur Proteins. Chem. Rev. 2004, 104, (2), 527-560. Lippard, S. J.; Berg, J. M., Principles of Biainarganic Chemistry. University Science Books: Mill Valley, CA, 1994. Gutlich, P.; Link, R.; Trautwein, A., Massbauer Spectrscapy and Transition Metal Chemistry. Springer - Verlag: Bertin, 1979. Rebeil, R.; Nicholson, W. L., The subunit structure and catalytic mechanism of the Bacillus subtilis DNA repair enzyme spore photoproduct lyase. Proceedings of the National Academy of Sciences of the United States of America 2001, 98, (16), 9038-43. Broderick, J. B.; Henshaw, T. F.; Cheek, J.; Wojtuszewski, K.; Smith, S. R.; Trojan, M. R.; McGhan, R. M.; Kopf, A.; Kibbey, M.; Broderick, W. E., Pyruvate fonnate-lyase-activating enzyme: Strictly anaerobic isolation yields active enzyme containing a [3Fe-4$]+ cluster. Biochemical and Biophysical Research Communications 2000, 269, (2), 451-456. Ollagnier de-Choudens, S.; Fonetcave, M., The lipoate synthase from Escherichia coli is an iron-sulfur protein. FEBS Letters 1999, 453, 25-28. Buis, J. M.; Cheek, J.; Kalliri, E.; Broderick, J. B., Characterization fo an active spore photoproduct lyase, a DNA repair enzyme in the radical sam superfamily. Journal of Biological Chemistry 2006, In Press. Henshaw, T. F.; Cheek, J.; Broderick, J. B., The [4Fe-4S]1+ Cluster of Pyruvate Formate-Lyase Activating Enzyme Generates the Glycyl Radical on Pyruvate Formate-Lyase: EPR-Detected Single Turnover. Journal of the American Chemical Society 2000, 122, (34), 8331-8332. Lieder, K. W.; Booker, S.; Ruzicka, F. J.; Beinert, H.; Reed, G. H.; Frey, P. A., S-Adenosylmethionine-Dependent Reduction of Lysine 2,3- Aminomutase and Observation of the Catalytically Functional Iron-Sulfur Centers by Electron Paramagnetic Resonance. Biochemistry 1998, 37, (8), 2578-2585. 86 12. 13. 14. Cheek, J.; Broderick, J. B., Adenosylmethionine-dependent iron-sulfur enzymes: versatile clusters in a radical new role. J. Biol. Inorg. Chem. 2001, 6, (3), 209-26. Jarrett, J. T. The novel structure and chemistry of iron-sulfur cluster in the adenosylmethionie—dependent radical enzyme biotin synthase. Archives of Biochemistry and Biophysics 2003, 433, 312-321. Broderick, J. B., Iron-sulfur clusters in enzyme catalysis. Comprehensive Coordination Chemistry II 2004, 8, 739-757. 87 CHAPTER V ENZYMATIC ACTIVITY OF SPORE PHOTOPRODUCT LYASE V.1 Introduction In the late 19th century it was discovered that a number of bacterial species spend part of their lives in a dormant cellular structure known as endospores.1 These endospores are some of the hardiest forms of life on the planet. Endospores exhibit extreme resistance to desiccation, harsh chemicals, heat, UV radiation and y radiation. Much effort has been expended investigating the molecular mechanism responsible for this high resistance to harsh environments. Some of the factors affecting resistance include the spare coat, the core water content, mineral content, the presence of SASPS and an ability to repair macromolecules such as DNA. The latter is the focus of this work. UV irradiation of spores has been shown to induce a specific type of DNA damage, known as the spare photoproductz’ 3 Spore photoproduct lyase is known to repair the DNA lesion spore photoproduct.4 Most likely this occurs immediately after the onset of spare germination since little metabolic activity occurs in the dormant spore phase.5' 6 Although it has been shown that spore photoproduct lyase can repair DNA 88 damage both in viva and in vitro, there had been no published specific activity of the spare photoproduct lyase nor has repair been correlated to the amount and type of iron sulfur cluster present in the protein. Spore photoproduct has been generated in viva upon exposure of sporulating bacteria such as B. subtilis or non-sporulating bacteria overexpressing SASP proteins to UV light.7 Spore photoproduct has also been produced in vitro by the addition of SASP to DNA followed by UV irradiation.8 SP 9,10 has also been synthesized in the laboratory, although not incorporated into a DNA oligomer. In all cases, the structure of SP has been determined/verified by 3,7,8,10,11 NMR techniques. Repair by spore photoproduct lyase has been seen in all of the above circumstances. Due to difficulty in working with bacterial spores and difficulties in synthesizing SP, in this work we have produced SP by the irradiation of the radiolabeled (at thymine) pUC18 plasmid DNA under SP forming conditions. Upwards of 5% of the thymine is converted to SP using the methods described herein. Repair of SP can be monitored by HPLC separation of thymine and SP followed by liquid scintillation counting. Using these methods, we have examined SP repair by SPL as a function of time to acquire a specific activity for SPL. Repair of SP can be monitored by HPLC separation of thymine and SP and liquid scintillation counting. We have also examined the effect of the cluster redox state on SP repair activity. 89 v.2 Experimental methods Materials Except where noted otherwise, all chemicals were purchased commercially and were of high purity. Radiolabeled [methyl - 3I-l] thymidine was purchased from Amersham Biosciences. 3H labeling and synthesis of the spare photoproduct Labeling of pUC18 DNA was carried out as follows. NovaBlue E. coli (Novagen) carrying pUC18 was grown overnight at 37 °C in 50 mL of 2 x YT medium (per liter: 16 g tryptone, 10 g yeast extract, 5 9 NaCl) containing 50 pg/mL ampicillin, 0.45 mM 2’-deoxyadenosine, and 10 uM [methyl-3H]-thymidine (0.74 MBq/mL) (Amersham-Pharrnacia). Labeled DNA was extracted from the overnight culture using a Promega Wizard Mini-Prep Kit. The specific activity of the purified DNA was typically 1.5 x 107 cpm/umol. To form a complex between the 3H-DNA and the SASP-C, the two were mixed in a 5:1, SASP-C: DNA ratio in 70 pL of 25 mM Tris-acetate, 125 mM NaCI, pH 6.5 and incubated at 37 °C for 2 hours in a custom made quartz vacuum hydrolysis tube. This complex was irradiated with 30 kJ/m2 UV light at 254 nm in order to form spore photoproduct on the 3H-DNA. This 3H-DNA was then used in the assay. The amount of spare photoproduct generated was quantified by hydrolyzing the irradiated 3H-DNA and separating SP from thymine on a Waters Spherisorb SSP 4.0 X 250 mm analytical column, run with M0 H20 at a flowrate of 1.8 mL/min in degassed M0 90 H20 for 25 min with fractions collected every 0.5 or 1 min. Liquid scintillation counting was performed on each fraction using a Wallac 1414-001 Liquid Scintillation Counter. Labeled thymine elutes at ~ 3 min and the spare photoproduct elutes at 10-11 min. Dividing the CPM eluting of the SP peak by the total CPM eluting from the column gives the percentage of thymine in the spare photoproduct form. The total amount of SP present is calculated as follows: [A260 DNA*50,ug/mLJ*1325 T * SPCPM Amount of SP = 1700 kDa pUC18 Total T CPM (1) Time course repair assay of spare photoproduct SP lyase activity was determined using a slightly modified version of the assay developed by Nicholson and coworkers. All solutions except DNA and protein solutions were prepared anaerobically in an mBraun glove box just prior to use. Protein and DNA solutions were made anaerobic by repeated vacuum- purge cycles prior to bringing them into the glove box. Assay experiments with and without SP lyase (1 uM) were set up in parallel in custom-made vacuum hydrolysis tubes in the glove box. Reaction mixtures (1 mL total volume) contained 1 nmol SP lyase, 2 mM AdoMet, 25000-50000 cpm of 3H-pUC18 DNA (3H at the thymine methyl and containing 200-435 nmol SP), 4 mM dithiothreitol, 3 mM dithionite, and 30 mM KCI, all in 20 mM sodium phosphate, 500 mM NaCl, 5% glycerol, pH 7.0. All reaction mixtures were incubated under anaerobic conditions at 37 °C for 60 minutes with aliquots taken every 10 minutes and 91 terminated with the addition of 0.5 mL trifluoroacetic acid. The hydrolysis tubes were sealed under vacuum and heated to 165 °C for 2 hours. The trifluoracetic acid was evaporated and the dried residue was resuspended in 100 uL of M0 H2O. A portion of the solution (150L) was loaded onto a Waters Spherisorb S5P 4.0 X 250mm analytical column and run at a flow rate of 1.8mUmin in degassed MQ H20 for 25 min with fractions collected every 0.5 or 1min. Liquid scintillation counting was performed on each fraction using a either a Wallac 1414-001 Liquid Scintillation Counter or a Beckman LS 6500 Liquid Scintillation Counter with the addition of 15 mL of Safety Solve liquid scintillation cocktail. Repair assay with pre-reduced SPL Time course repair assays were carried out as above except that SPL used had previously been reduced under anaerobic conditions by the addition of 10 mM sodium dithionite and 10 mM DTT. EPR spectroscopy was conducted on a 300 pL sample of SPL that was 550 uM in concentration on an X-band Bruker ESP300E spectrometer equipped with a liquid He cryostat and a temperature controller from Oxford Instruments. Spectra were recorded at 12 K. Spin quantifications were carried out as previously described. The double integrals of the EPR signals were evaluated by using a computer on-line with the spectrometer. Spin concentration in the protein samples was determined by calibrating double integrals of the EPR recorded under non-saturating conditions with a standard sample of 0.1 mM Cu (II) and 1 mM EDTA. 92 V.3 Results and discussion After purification and irradiation of pUC18 methyl 3H thymidine DNA, the amount of spare photoproduct produced as a total percentage of thymine was between 4 and 7% (Figure V.1). 500 400 — 300 cpm 200 100 -, min Figure V.1 The separation of thymine and SP after HPLC is illustrated above. The solid line is UV-irradiated and hydrolyzed DNA without the addition of SPL. The dashed line is UV-irradiated and hydrolyzed DNA after the addition of SPL and incubated for 60 min. Both samples were acid hydrolyzed and loaded onto a Waters Spherisorb S5P column and run with an isocratic flow of degassed MQ water for 25 minutes at 1.8 mLImin. Fractions were collected every minute and run on a liquid scintillation counter. This DNA proved to be stable under repair conditions in the absence of SPL, after 1 hour incubation at 37°C, no change in the amount of SP was seen. Time course experiments show the addition of SPL to SP under reducing conditions 93 causes a reduction in the amount of spare photoproduct present. This repair is linear over the course of 1 hour (Figures V2 and V3) with 70% of the SP repaired after 1 hour. Prior to our work, repair of SP by SPL has been reported in I No SPL I No SPL ~~ I10mm 2000-/ I 20 min a 30 min III 40 min I 50 min CI 60 min 30001/ * cpm 1000: . . _ ,_ N 31 Increasing elution time (min) N Time (0. 60min) Figure v.2 A representation of SP repair over a time of 60 min. Thymine elutes at ~ 3 min, and SP elutes at ~10 min. Over the course of 60 minutes the amount of SP decreases 70%. terms of the percentage of SP repaired. While useful in illustrating the presence of SP repair activity, numbers in terms of % SP repaired are not standard activity rates do not allow for any sophisticated enzyme kinetics. By plotting of nmols of SP repaired versus time, in contrast a standard specific activity can be calculated 94 (Figure V3). The specific activity of SPL based on our work to date is 0.33 pmols SP repaired/min/mg SPL.12 o A 00 N l J pmol SP repaired .0 h O time(min) Figure v.3 SP lyase is active in SP repair. Representative time course of SP repair by reduced SP lyase. Repair of pUC18 DNA was monitored at 10 min intervals by removal of 100 pL aliquots, which were quenched hydrolyzed, and monitored by HPLC for SP repair. Linear repair is observed up to 60 minutes with a specific activity of 0.33 pg SP repaired/minlmg SPL. The apparent lag time may result from a need for reduction of SP lyase prior to the initiation of SP repair.12 While the time course repair assay shows that a specific activity can be calculated for SPL, the calculation did not take into account the degree to which SPL was in the [4Fe-4S]1+ state. It is has been shown in other members of the radical SAM superfamily such as PFL-AE and LAM that the [4Fe-4S]1+ is the active cluster form. It is therefore important to determine a specific activity based on the amount of [4Fe-4S]1+ cluster present in reduced SPL. To this end, SPL 95 that had been previously reduced with sodium dithionite and whose cluster content had been measured by EPR spin quantification (Figure V.4) was used in a repair assay. EPR Intensity 2800 3000 3200 3400 3600 3800 4000 Field (Gauss) Figure v.4 EPR of SPL used in repair of SP dimers shows that 54% of the protein is in the [4Fe-4S]1+ state, as measured by spin quantification versus a Cu-EDTA standard, prior to its assay for DNA repair activity. The SPL contained 54% [4Fe-4S]1+ (297 pM) and repair assays run with this protein show linear repair up to 50 minutes (Figure v.5). A specific activity of 1.33 pmols SP repaired/minlpg SPL was calculated, which if calculated by content of [4Fe—4S]1+ present is 2.4 umols SP repaired/min/mg SPL with [4Fe-48]1+. This is over a 4 fold increase in activity compared with protein that was not reduced prior 96 A. 100001 9%, mm— ‘ lwmn l D40min I30min CPM 5000— “Om," I 10 min 2500‘ I blank 0- \b \' ‘\ elution time (min) 21 Increasing time B 0.8 0.6 - .0 2 '5 O. 2 a. 0.4 ~ in '6 E 3 0.2 ~ 0 I I T I F 0 10 20 30 40 50 60 Time (min) Figure V.5 Time course of SP repair by reduced SPL. A. Representative HPLC chromatograms show loss of the SP peak as a function of time. B. Linear repair is observed up to 50 minutes with a specific activity of 1.33 umol SP repaired/min/mg SPL. 97 to use in SP repair assays, suggesting that in previous assays the rate was being limited by the amount of [4Fe-4S]1+ cluster present. The amount of reductant in those assays may have been insufficient for complete cluster reduction. By reducing prior to DNA repair, a greater percentage of the protein is active. v.4 Conclusions Prior reports as to the DNA repair capabilities of SPL have provided activity in terms of % DNA repaired and have not mentioned a specific activity of the enzyme.” 14 We were however able to calculate a specific activity of 0.0002 pmol SP repaired/min/mg SPL based on the information in the literature, nearly 1000 fold lower than our activity reported here.14 The determination of an accurate specific activity is important to future studies involving the kinetics and mechanism of SPL. In addition, our results indicate that SPL is much faster at SP repair than initially thought, which may be important as SPL is thought to be active only during cell germination. That is, it is cnrcial for cellular function that SPL repairs the damaged DNA expediently in order for other proteins to be produced correctly. We have also shown that the activity of SPL can be enhanced by reducing the protein prior to use in repair assays. In the “standard” assay described in this chapter, as-isolated SPL is added immediately to a SP repair reaction with 3 mM sodium dithionite present. We then start timing the SP repair and remove samples every 10 minutes. This method leads to a specific activity of 0.33 pmol SP repaired/min/mg SPL, however, plots of SP repaired versus time show a an 98 amount of lag time present in SP repair and are not fully linear. This may be the result of the protein not being reduced to a [4Fe-4S]1+ cluster at the beginning of the reaction, thus inhibiting SPL’s repair rate. By chemically pre-reducing the SPL, our activity increased 4 fold to ~1.33 pmol SP repaired/min/mg SPL. By correlating this activity to the amount of protein that contains a [4Fe-4S]1+ cluster based on EPR we obtain an activity of 2.4 pmol SP repaired/min/mg SPL with [4Fe-4S]1+. Based on this evidence of enhanced activity with pre-reduced SPL, we conclude that similar to other members of the radical SAM superfamily15'16, SPL utilizes a [4Fe-4S]1+ as the active cluster. Confirmation of this conclusion will require a more detailed study examining the correlation between the amount of [4Fe-4S]1+ cluster present in the protein and the rate of SP repair. 99 V.5 10. 11. References Cohn, F., Untersuchungen uber Bakterien IV. Beitrage zur Biologie der Bacillen. Beitre. Biol. Pflanz 1876, 2, 249-276. Donnellan, J. E., Jr.; Setlow, R. B., Thymine photoproducts but not thymine dimers found in ultraviolet-irradiated bacterial spores. Science 1965, 149, 308-310. Donnellan, J. E., Jr.; Stafford, R. S., Ultraviolet photochemistry and photobiology of vegetative cells and spores of Bacillus megaterium. Biophysical Journal 1968, 8, (1), 17-28. Munakata, N.; Rupert, C. 8., Dark repair of DNA containing spore photoproduct in Bacillus subtilis. Molecular and General Genetics 1974, 130, (3), 239-50. Setlow, P., I will survive: protecting and repairing spore DNA. J. Bacterial. 1992, 174, 2737-2741. Setlow, P., Resistance of Spores of Bacillus Species to Ultraviolet Light. Environmental and Molecular Mutagenesis 2001 , 38, 97-104. Setlow, B.; Hand, A. R.; Setlow, P., Synthesis of a Bacillus subtilis small, acid-soluble spore protein in Escherichia coli causes cell DNA to assume some characteristics of spare DNA. Journal of Bacteriology 1991, 173, (5), 1642-53. Nicholson, W. L.; Setlow, B.; Setlow, P., Ultraviolet irradiation of DNA complexed with alpha/beta-type small, acid-soluble proteins from spores of Bacillus or Clostridium species makes spore photoproduct but not thymine dimers. Proceedings of the National Academy of Sciences of the United States of America 1991, 88, (19), 8288-92. Nicewonger, R.; Begley, T. P., Synthesis of the spore photoproduct. Tetrahedron Letters 1997, 38, (6), 935-936. Friedel, M. G.; Berteau, 0.; Pieck, J. C.; Atta, M.; Ollagnier-de-Choudens, S.; Fontecave, M.; Carell, T., The spore photoproduct lyase repairs the 5S- and not the 5R-configured spore photoproduct DNA lesion. Chemical Communications (Cambridge, United Kingdom) 2006, (4), 445-447. Chandor, A.; Berteau, 0.; Douki, T.; Gasparutto, D.; Sanakls, Y.; Ollagnier-de-Choudens, S.; Atta, M.; Fontecave, M., Dinucleatide spore photoproduct: A minimal substrate of the DNA repair spore photoproduct lyase enzyme from bacillus subtilis. J. Biol. Chem. 2006, In Press. 100 12. 13. 14. 15. 16. Buis, J. M.; Cheek, J.; Kalliri, E.; Broderick, J. B., Characterization fo an active spore photoproduct lyase, a DNA repair enzyme in the radical sam superfamily. Journal of Biological Chemistry 2006, In Press. Rebeil, R.; Sun, Y.; Chooback, L.; Pedraza-Reyes, M.; Kinsland, 0.; Begley, T. P.; Nicholson, W. L., Spore photoproduct lyase from Bacillus subtilis spores is a novel iron-sulfur DNA repair enzyme which shares features with proteins such as class III anaerobic ribonucleotide reductases and pyruvate-formate lyases. Journal of Bacteriology 1998, 180, (18), 4879-85. Slieman, T. A.; Rebeil, R.; Nicholson, W. L., Spore photoproduct (SP) lyase from Bacillus subtilis specifically binds to and cleaves SP (5- thyminyl-5,6—dihydrothymine) but not cyclobutane pyrimidine dimers in UV- irradiated DNA. Journal of Bacteriology 2000, 182, (22), 6412-7. Henshaw, T. F.; Cheek, J.; Broderick, J. B., The [4Fe-4$]1+ Cluster of Pyruvate Formate-Lyase Activating Enzyme Generates the Glycyl Radical on Pyruvate Formate-Lyase: EPR-Detected Single Turnover. Journal of the American Chemical Society 2000, 122, (34), 8331-8332. Gambarelli, S.; Luttringer, F.; Padovani, D.; Mulliez, E.; Fontecave, M., Activation of the anaerobic ribonucleotide reductase by S- adenosylmethionine. ChemBiaChem 2005, 6, (11), 1960-1962. 101 CHAPTER VI MECHANISTIC CONSIDERATIONS FOR SPORE PHOTOPRODUCT LYASE Vl.1 Introduction S-adenosylmethionine has been shown to act as both a co-factor and a co-substrate among members of the radical SAM superfamily.1 For example, in PFL-AE and anRNR-AE a single SAM is used to activate a single PFL or anRNR by generation of a glycyl radical.” 2 ln LAM, however, one SAM molecule can lead to multiple enzyme turnovers.3 Whether the subtle difference observed between PFL-AE and LAM by ENDOR and EXAFS reflect this difference in utilization of SAM remains to be detennined.“ There is still debate as to whether SAM acts as either a cofactor or as a cosubstrate with SPL. A mechanism originally proposed by Mehl and Begley (Scheme Vl.1) gives SAM the role as an enzyme cofactor.8 In this mechanism, an electron from presumably the [4Fe-4S]1+ cluster of SPL is transferred to SAM causing reductive cleavage to form the putative 5’-deoxyadenosyl radical and methionine. The 5’-deoxyadenosyl radical initiates radical catalysis by abstracting a H atom from the C-6 position of the thymine ring. This initial H atom abstraction 102 H 3_NHO sugar——N O sugar—N phasp\hate phosphate H CH2 0:5 .H \SUQaF—Nkéz-i-fl sugar—N 0 +8 ’ A g NH3+ HO OH NH3+ TT SP 0 H )—N /SU93F—N, H___r“’-":==O /sugar——N _ o phosph/ate phosphate H CH2 \ 0 ,H ) ):NH sugar—+1 sugar—N o H H CH3 H . CH3 :4 H A Cth e H 0 +IS ' A $113 S sugar—M’_ NH 0 sugar—N phosphate H CH3 phosphate 0 ,H \ )—~ sugar—N __ *0 .— sugar—N H CH3 -Ooc~(\/CH3I :;0 jH -OOCW NH3+ NH3+ Scheme Vl.1 Proposed mechanism for SP repair by SPL in which SAM is homolytically cleaved by an electron from the iron sulfur cluster of SPL. A putative 5’-deoxyadenosyl radical is formed which abstracts a hydrogen from the C-6 on the thymine ring and initiates radical catalysis.8 103 has been supported by the work of Cheek and Broderick.9 After H atom abstraction by 5’-deoxyadenosine, a radical mediated B-scission cleaves the methylene bridge and separates the thymine bases, leaving a methyl radical on one of the thymine bases. This methyl radical then abstracts a H atom from 5’- deoxyadenosine to reform the putative 5’-deoxyadenosyl radical. This radical is then free to interact with methionine and reform SAM donating its electron back to the FeS cluster. However, work in the Nicholoson lab and the Fontecave lab show cleavage of SAM into 5’-deoxyadenosine and methionine by SPL both non- 11,12 productively1O and during repair of model substrates. As mentioned above though, work carried out in the Broderick group confirmed part of Mehl and Begley’s mechanism by showing 3H label transfer from the C-6 of the thymine ring to SAM during repair.9 This helped to substantiate the proposed mechanism in Scheme Vl.1 involving SAM as a co-factor in SPL catalysis. A recent DFT study also provided evidence that the proposed mechanism involving SAM as a cofactor was energetically plausible.13 This study concluded the abstraction of a hydrogen atom from 5’-deoxyadenosine should be the limiting step in this reaction with an energy barrier of 23.1 kcal/mol. The goal of the work described in this chapter was to further investigate the mechanism of SP repair by SPL with a particular focus on the role of SAM. If SAM acts as a cofactor, then no 5’-deoxyadenosine should be formed during repair. We have therefore conducted experiments in which we monitor the production of 5’-deoxyadenosine and the loss of SAM during repair of SP. Based on the mechanism proposed in Scheme Vl.1 if SAM is labeled at the 5-C position 104 with 3H (Scheme Vl.2), a portion of this should be transferred to thymine during enzymatic turnover. This would occur when the methyl radical abstract a hydogren from 5’-deoxyadenosine to reform the putative 5’-deoxyadenosyl radical. The transfer of this label from SAM to thymine should be subject to a selective isotope effect, as the hydrogen on the methyl group of 5’-dAdo would be energetically favored for abstraction versus tritium. CH3 U +8 -OOC NH3+ HO OH Scheme Vl.2 S-adenosylmethionine with a tritium label at the 5’-C. /sugath)—v :___'\‘H:C::) phosph95% of the 7 nmols of AdoMet present in the reaction remains as AdoMet. Similar results are obtained after overnight incubation (Figure Vl.1 D). The data obtained from these chromatograms indicating the amount of 5’-dAdo produced and the amount of SP repaired is summarized in Table Vl.1. The results clearly indicate that AdoMet is Sample Time of SP repaired 5’-dAdo Specific Activity of incubation (nmol) produced SPL (Hours) (nmol) (pmol SP repaired/minlmg) B 24 0 0 N/A C 1 .5 146 0.1 0.16 D 24 150 0.03 0.0104 Table Vl.1 Summary of SAM cleavage during SP repair reaction. not cleaved stoichiometrically to methionine and 5’-deoxyadenosine during SP repair. The lack of AdoMet cleavage observed here differentiates our results from two previously published reports, which show 5’ deoxyadenosine production by SPL;1O'11 the reasons for these differences remains unclear although they could be an artifact of cluster reconstitution in the other studies. The results above have been published in the Journal of Biological Chemistry.18 113 Direct H atom transfer ham [2, 5 ’, 8 — 3H]-SAM to repaired thymine SAM, labeled with 3H at the 5’ position (Scheme Vl.2) was successfully synthesized and purified via cation exchange chromatography to yield of 34%. We have previously shown that SP repair is initiated by direct H-atom abstraction from the C-6 position of SP by an AdoMet-derived 5’deoxyadenosyl radical intermediate.9 These results support a mechanism (Scheme Vl.1) in which the resulting substrate radical undergoes a radical mediated B-scission to generate the product thymine radical. The product thymine radical is then proposed to re- abstract a H atom from the 5’-deoxyadenosine formed in the first step, thereby re-generating the 5’-deoxyadenosyl radical intermediate. The 5’-deoxyadenosyl radical could then recombine with methionine to reform AdoMet as Shown in Scheme Vl.1. In order to further investigate this mechanism, we have carried out repair assays in which the AdoMet is labeled at the 5’-position with tritium. The mechanism shown in Scheme Vl.1 would predict a label transfer from this position into the repaired thymine. Repair assays were carried out using the labeled AdoMet and unlabeled irradiated DNA. Control experiments were conducted in the absence of SPL to check for erroneous label transfer or other experimental problems. In all cases, repaired DNA was isolated from the other reaction components, hydrolyzed and then subjected to chromatography on a Waters Spherisorb S5P column. Analysis of the fractions by scintillation counting reveals a peak at 3 minutes (Figure Vl.2), the elution time of thymine under these 114 conditions. This peak is observed reproducibly in experimental samples containing SPL and is absent in samples containing no SPL. Furthermore, the amount of label appearing in the thymine peak is consistent with what we would predict (115 CPM) based on the Specific activity of the starting AdoMet and using 15.8 as an estimate of the tritium Time (min) Figure Vl.2 3H transfer from labeled [2, 5’, 8 — 3Hj-SAM to repaired thymine. DNA repaired by SP lyase in the presence of [2, 5’, 8 — 3Hj-SAM (solid lines are duplicate experiments) shows 3H incorporation into thymine (elution time of 3 min.). Samples prepared in the absence of SPL Shows no such incorporation (dashed lines).18 isotope effect.14 This calculation was done as follows: theoretically, 7.3 nmol of SP was repaired in this reaction, as SAM (70 nmol) was added in excess, we thus assume that 7.3 nmol of SAM reacted during the course of SP repair. The 115 activity of the [2, 5', 8 - 3Hj-SAM used was 1,500,000 DPM/ pmol SAM or 750,000 CPM/pmol SAM, however only 1/3 of this activity is present at the 5’ position giving 250,000 CPM/pmol 5’ 3H. In our reaction if 7.3 nmol of SAM was used, then the theoretical value for the CPM expected after label transfer would be 1875 CPM. However, there should be an isotope effect as to which of the three H atoms from the methyl group of 5’-dAdo will be abstracted. We have used the estimate of 15.8 for the isotope effect, which gives us a theoretical value of 115 CPM. After subtracting the background CPM from our results in Figure Vl.2, the CPM observed in the thymine peaks are 124 and 100. This is close to our theoretical value and well within experimental error. The transfer of the expected amount of tritium from AdoMet to thymine during repair by SPL provides further support for a mechanism in which AdoMet is a catalytic cofactor, and in which there is direct H atom transfer between AdoMet—derived deoxyadenosyl radicals and substrate product. The results reported above have been published in the Journal of Biological Chemistry.18 A potential isotope effect for H atom abstraction AS mentioned above, previous studies of SPL had shown evidence for direct H atom abstraction from the C-6 position of SP by the putative 5’- deoxyadenosyl radical.9 In these studies, SP was produced in which the 06 position of thymine was labeled with tritium and transfer to AdoMet was observed, however this study did not attempt to quantify the tritium transfer or examine its rate. By quantifying tritium transfer at various extents of reaction, we 116 can determine the tritium isotope effect on the overall reaction from the initial H atom abstraction. Further, by comparing the theoretical amount of tritium transferred after complete reaction to our experimental value, we can determine whether the initial H-atom abstraction is stereospecific. For example if SP is created non-stereospecifically and if SPL stereospecifically abstracts a H atom from C6 then the CPM observed should be equal to 1/2 the theoretical amount of label transfer possible given the amount of SP used. On the other hand if SPL is non-stereospecific, then the amount of label transfer would be less because of the preference for abstracting an H atom over a tritium atom. Other possibilities include the case of SP production being stereospecific, if it is stereospecific and SP is stereospecific, then we would observe either no label transfer or 100% label transfer depending upon the stereospecificity involved. In order to address these questions, we used a defined amount of SP (311 nmol) tritiated at C-6. The amount of time the assays were incubated was determined by use of the previously determined specific activity for SPL (0.33pmol/min/mg SPL). In order to repair 311 nmol of SP with ~0.1mg of SPL, 10 minutes is required to repair 100% of the SP lesions. The calculated activity of the SP is ~1,000 CPM/ 1 nmols SP, thus after 100% repair of ~311 nmol of SP, 7500 CPM should be transferred to SAM if the SPL repair reaction is stereospecific and creation of SP is non-stereospecific; for 50% SP repair, ~3770 CPM Should be present in SAM and at 25% SP repair ~1880 CPM. However, if there is a tritium isotope effect of 15.8, the 50% and 25% SP repair samples should only have 238 and 119 CPM. 117 Our results closely match these values above as shown in Figure Vl.3 and summarized in Table Vl.2. At full reaction 5928 CPM is observed (78% of the theoretical value) and the DNA is fully repaired. Due to the complex experimental techniques involved, it is likely that certain % of the label is lost during the experiment. As such we have used the 100% SP repair reaction as our standard 500 5928 CPM CCU-N0 SPL 6: Reaction 25% 400 ‘ IReaction 50% IReaction 100% 300 s E n. U 200 ~ :5 100 d L“. '3 :3 , O _. i , ., . H . .. H .-.J - Wt 1 2 3 4 5 6 7 8 9 10 Time (min) Figure Vl.3 Tritium isotope effect for H atom abstraction. The bar graph above shows the number of counts present in purified SAM after use in a SP repair reaction with [C-6 - 3H]-thymine SP. Reaction times shown are for 0, 2.5, 5 and 10 minutes. An isotope affect between 15.1 and 17.2 can be calculated for tritium during SP repair and H atom abstraction. 118 to determine what % of CPM (22%) is lost during the procedure. At 50% repair we observe the CPM to be 172, by using the 100% reaction as a correction factor we would obtain 1720PM/0.78 = 220 CPM. An isotope effect can then be calculated by dividing the theoretical amount of CPM given no isotope effect by the experimentally determined CPM (220CPM) to give a value of 17.2. The same can be done at 25% reaction, giving a value of 15.1. Sample % of SP Theoretical Theoretical Experimental Calculated repaired Label Label Label transfer isotope transfer with transfer with (CPM) effect no isotope and isotope effect effect of 15.8 (CPM) (CPM) 1 0 0 0 N/A 2 25 1890 1 1 9 99 1 5.1 3 50 3770 238 172 1 7.2 4 1 00 7540 7535 5928 N/A Table Vl.2 Summary of label transfer from C-6 to SAM at increasing SPL repair reaction times and the corresponding isotope effect. The results above give us insight into the reaction of SPL. First, these results indicate that SP formation is non-stereospecific while, SP repair by SPL is stereospecific, though the result in no way indicate which C-6 H atom is abstracted, only that it is abstracted stereospecifically. Also these results indicated the presence of a tritium isotope effect for initial H atom abstraction, suggesting that H atom abstraction from the C-6 is rate determining. 119 Vl.4 Conclusions Our observation of 3H Label transfer from SAM to repaired thymine is supports Mehl and Begley’s suggested mechanism for SP repair (Scheme Vl.1).8 This mechanism involves initial SAM cleavage by electron donation from the iron sulfur cluster to form the putative 5’-deoxyadenosyl radical. This radical species has been shown abstract a hydrogen from the C-6 position of the thymine ring, resulting in label transfer SAM. The mechanism proposes that the C-6 hydrogen is abstracted leaving an organic radical at C6 The SP then undergoes a radical- mediated B-scission resulting in the separation of the adjacent thymine bases. The resulting methyl radical abstracts hydrogen from 5’-deoxyadenosine to reform the putative adenosyl radical, which can reform SAM by electron donation to the iron sulfur cluster and go on to repair further spore photoproducts. In the last step of the step of SP repair, a hydrogen from 5’deoxyadenosine is abstracted and transferred to repaired thymine. If this mechanism is accurate, then labeling the 5’C of SAM with tritium Should result in label transfer to SAM. Our work confirms that indeed this 5’ label is transferred from SAM to repaired thymine. This evidence together with other evidence that Show that a single SAM can be used to repair over 400 SP lesions and that the production of 5’-deoxyadenosine is not observed during repair provides strong evidence for SAM’s role as a cofactor in the repair of SP by SPL. It also strongly supports the mechanism proposed in Scheme Vl.1. 120 The observation of a tritium isotope effect of ~15-17 observed during initial H atom abstraction suggests that this step is energetically high and may be the rate limiting step of the reaction. This would be in conflict with DFT calculations that predict an energy barrier of 14.1 kcallmol, far from the highest energy barrier calculated during the reaction.13 Rather, DFI' predicts that the production of the 5’-dAdo radical by the methyl radical is far more energetically unfavorable with a barrier of 23.1 kcallmol as the 5’-dAdo radical is predicted to be less stable then the thymine radical.13 These calculations, however, do not take into account the surrounding protein environment which can have a large effect on energy barriers. 121 V|.5 References Henshaw, T. F .; Cheek, J.; Broderick, J. B., The [4Fe-4$]1+ Cluster of Pyruvate Formate-Lyase Activating Enzyme Generates the Glycyl Radical on Pyruvate Formats-Lyase: EPR-Detected Single Turnover. Journal of the American Chemical Society 2000, 122, (34), 8331-8332. Harder, J.; Eliasson, R.; Pontis, E.; Ballinger, M. D.; Relchard, P., Activation of the anaerobic ribonucleotide reductase from Escherichia coli by S-adenosylmethionine. Journal of Biological Chemistry 1992, 267, (35), 25548-52. Moss, M.; Frey, P. A., The role of S-adenosylmethionine in the lysine 2,3- aminomutase reaction. Journal of Biological Chemistry 1987, 262, (31), 14859-62. Chen, D.; Walsby, C.; Hoffman, B. M.; Frey, P. A., Coordination and Mechanism of Reversible Cleavage of S-Adenosylmethionine by the [4Fe- 481 Center in Lysine 2,3-Aminomutase. Journal of the American Chemical Society 2003, 125, (39), 1 1788-1 1789. Walsby, C. J.; Ortillo, D.; Broderick, W. E.; Broderick, J. B.; Hoffman, B. M., An Anchoring Role for FeS Clusters: Chelation of the Amino Acid Moiety of S-Adenosylmethionine to the Unique Iron Site of the [4Fe-48] Cluster of Pyruvate Formate-Lyase Activating Enzyme. Journal of the American Chemical Society 2002, 124, (38), 1 1270-1 1271 . Walsby, C. J.; Hong, W.; Broderick, W. E.; Cheek, J.; Ortillo, D.; Broderick, J. B.; Hoffman, B. M., Electron-Nuclear Double Resonance Spectroscopic Evidence That S-Adenosylmethionine Binds in Contact with the Catalytically Active [4Fe-4S]+ Cluster of Pyruvate Formate-Lyase Activating Enzyme. Journal of the American Chemical Society 2002, 124, (12), 3143-3151. Walsby, C. J.; Ortillo, D.; Yang, J.; Nnyepi, M. R.; Broderick, W. E.; Hoffman, B. M.; Broderick, J. B., Spectroscopic Approaches to Elucidating Novel Iron-Sulfur Chemistry in the \"Radical-SAMV' Protein Superfamily. Inorganic Chemistry 2005, 44, (4), 727-741. Mehl, R. A.; Begley, T. P., Mechanistic Studies on the Repair of a Novel DNA Photolesion: The Spore Photoproduct. Organic Letters 1999, 1, (7), 1065-1066. Cheek, J.; Broderick, J. B., Direct H Atom Abstraction from Spore Photoproduct C-6 Initiates DNA Repair in the Reaction Catalyzed by Spore Photoproduct Lyase: Evidence for a Reversibly Generated Adenosyl Radical Intermediate. Journal of the American Chemical Society 2002, 124, (12), 2860-2861. 122 10. 11. 12. 13. 14. 15. 16. 17. 18. Rebeil, R.; Nicholson, W. L., The subunit structure and catalytic mechanism of the Bacillus subtilis DNA repair enzyme Spore photoproduct lyase. Proceedings of the National Academy of Sciences of the United States of America 2001, 98, (16), 9038-43. Friedel, M. G.; Berteau, O.; Pieck, J. C.; Atta, M.; Ollagnier-de-Choudens, S.; Fontecave, M.; Carell, T., The spore photoproduct lyase repairs the SS- and not the 5R-configured spore photoproduct DNA lesion. Chemical Communications 2006, (4), 445-447. Chandor, A.; Berteau, O.; Douki, T.; Gasparutto, D.; Sanakis, Y.; Ollagnier-de-Choudens, S.; Atta, M.; Fontecave, M., Dinucleotide spore photoproduct: A minimal substrate of the DNA repair Spore photoproduct lyase enzyme from bacillus subtilis. J. Biol. Chem. 2006, In Press. Guo, J.-D.; Luo, Y.; Himo, F., DNA repair by spore photoproduct lyase: a density functional theory study. Journal of Physical Chemistry B 2003, 107, (40), 11188-11192. O'Leary, M. H., Multiple isotope effects on enzyme-catalyzed reactions. Annual Review of Biochemistry 1989, 58, 377-401 . Chih, H. W.; Marsh, E. N. G., Tritium partioning and isotope effects in adenosylcobalamin-dependent glutamate mutase. Biochemistry 2001, 40, (13060-13067). Sun, Y.; Palasingam, K.; Nicholson, W. L., High-pressure liquid chromatography assay for quantitatively monitoring spore photoproduct repair mediated by spore photoproduct lyase during germination of uv- irradiated Bacillus subtilis spores. Analytical biochemistry 1994, 221, (1), 61-5. Rebeil, R.; Sun, Y.; Chooback, L.; Pedraza-Reyes, M.; Kinsland, C.; Begley, T. P.; Nicholson, W. L., Spore photoproduct lyase from Bacillus subtilis spores is a novel iron-sulfur DNA repair enzyme which shares features with proteins such as class III anaerobic ribonucleotide red uctases and pyruvate-formate lyases. Journal of bacteriology 1 998, 180, (18), 4879-85. Buis, J. M.; Cheek, J.; Kalliri, E.; Broderick, J. B., Characterization fo an active spore photoproduct lyase, a DNA repair enzyme in the radical sam superfamily. Journal of Biological Chemistry 2006, In Press. 123 CHAPTER VII DNA BINDING PROPERTIES OF SPORE PHOTOPRODUCT LYASE Vl|.1 Introduction A variety of proteins have been shown to interact and bind to DNA. Some of the more well studied DNA binding proteins include DNA photolyase,1 DNase I,2 EcoR1 endonuclease3 and topoisomerase I4 among a variety of others. The various DNA binding proteins have different methods of binding to the DNA and can be categorized into several families including helix-tum-helix,5 zinc binding, leucine zipper and beta hairpin/ribbon.6 As mentioned in the introduction, spore photoproduct lyase shares sequence homology with DNA photolyases towards its C-terrninal end.7 DNA photolyase is known to be a helix-tum-helix DNA binding protein and the region of sequence homology to SPL is in the vicinity of this binding motif.8 It is therefore reasonable to hypothesize that SPL utilizes a helix-tum-helix binding motif similar to that of DNA photolyase. There are many different ways to check for DNA-protein interactions, including DNA fingerprinting and gel-shift assays.6 The studies described herein 124 utilized the latter, to examine some of the DNA binding properties of SPL. The gel shift assay utilizes (usually) a 32F end-labeled DNA oligonucleotide, which can be electrophoresed in either the presence or absence of the target protein. If protein binds to the DNA, the oligonucleotide will be retarded on the gel and travel a shorter distance. By carrying out the experiment at a range of protein concentrations, a binding constant can be determined. We have used this technique to determine whether the presence or absence of an iron sulfur cluster or SAM affects the binding affinity of SPL to DNA. Previous studies in the Nicholson lab had demonstrated that SPL bound specifically to SP and repaired the DNA but did not bind to cyclobutane dimers or 6’4’- photoproducts.9 These studies, however, were not quantitative and thus did not provide dissociation constants. The previous studies also did not provide any insight as to what region of the protein bound to the DNA or if this binding was dependent upon the iron sulfur cluster of SPL or the co-factor SAM. For this reason we have used gel shift assays to look at the dissociation constant of SPL with DNA oligomers under varying conditions. Vll.2 Experimental Methods 32F End labeling of 94mer oligonucleotide Two complementary nucleotides were synthesized (Integrated DNA Technologies) based upon the B. subtilis sequence (322456-322550). These oligonucleotides are shown below: 125 5’-CGG GAT CAA CCA GAG CAT CAT GCT TGC G'l‘l' ATC AAT GGT TGT TAT CGC CGC AAT GGT CGG TGC ACC GGG ACT TGG TTC TGA AGT ATA CAG TGC C-3’ (TK4-72a) with the complementary strand being: 5’-GGC ACT GTA TAC TTC AGA ACC AAG TCC CGG TGC ACC GAC CAT TGC GGC GAT AAC AAC CAT TGA TAA CGC AAG CAT GAT GCT CTG G‘l'l’ GAT CCC G — 3’ (TK4-72b). The TK4-72a oligonucleotide (1 pL, 5.8 pmoles) was added to a 5 X Exchange reaction buffer (lnvitrogen) with 5 pL [7-32P] ATP (10 pCi/uL, 3000 Ci/mmol), 0.05 pL T4 polynucleotide kinase (lnvitrogen), and 13.5 pL autoclaved MQ water to final volume of 25 uL. The reaction mixture was incubated at 37 °C for 10 minutes, followed by heat inactivation at 65 °C for 10 minutes. The oligonucleotide was purified by standard ethanol precipitation procedures in which 250 uL of 4.67 M ammonium acetate and 100% ethanol (ice cold) were added. The oligomers were kept on ice for 30 minutes and centrifuged at 12,000 RPM for 20 minutes. The supernatant was removed and 500 pL of 80% ethanol was added followed by centrifugation at 12,000 RPM for 20 minutes. The supernatant was again removed and the oligomer was dried under nitrogen and resuspended in 50 uL TAE buffer. The labeled strand was annealed to the unlabeled complementary strand by adding 5.8 pmoles of the complementary strand and heating at 90 °C for 3 minutes then slowly cooling to room temperature over the course of 1 hour. The double stranded DNA oligo was 126 further purified using a Mini Spin Quick Oligo column (Roche Applied Science) and stored at -80 °C. Gel-shift DNA binding assay An 8% polyacrylamide gel was prepared using 10mL 40% acrylamide stock solution, 1 mL of 50 X TAE buffer (1 L contains 242 g Tris base, 57.1 mL glacial acetic acid, and 100 mL of 0.5 M EDTA pH 8.0), 0.35mL ammonium persulfate (0.1 g/mL) and 17.5 uL TEMED. The mixture was prepared and poured between glass plates to a final size of 20 cm x 20 cm x 1 mm. The gel was pre-electrophoresed at 100 V for 30 minutes before loading sample. Lane 1 2 3 4 5 6 7 9 10 11 Binding Buffer lid-L 2 2 2 2 2 2 2 2 2 2 50% Glycerol (9L) 94mer oligo (pmol) 0.46 0.46 0.46 0.46 0.46 0.46 0.46 0.46 0.46 0.46 0.46 NP-40 (le MQ H20 (le 11 10 SPL (pmol) 2.3 4.6 6.9 9.2 11.5 13.8 16.1 18.4 23 46 SPL (an 115 230 345 460 575 690 805 920 1150 2300 Protein to DNA Ratio 10 15 20 25 30 35 40 50 100 Table Vll.1 Reaction conditions for the gel shift assay of SPL and DNA under anaerobic conditions 127 Lane 10 11 Binding Buffer (le 50% Gyl (pL) 94mer oligo (pmol) 0.46 0.46 0.46 0.46 0.46 0.46 0.46 0.46 0.46 0.46 0.46 NP-40 (11L) MQ H20 (11L) 11 10 SPL (pmol) 4.6 9.2 13.8 18.4 23 27.6 32.2 36.8 46 92 SPL nM) 115 230 345 460 575 805 1 035 920 1 380 2300 Protein to DNA Ratio 10 15 20 25 35 45 60 100 200 Table Vll.2 Reaction conditions for the gel shift assay of SPL and DNA under aerobic conditions. 128 Lane 10 11 Binding Buffer (ill-L 50% Gly (11L) 94mer oligo (meL 0.11 0.11 NP-40 (HQ MQ H20 (uL) 11 10 SPL (pmol) 22 66 88 110 220 550 1100 1375 1650 SPL (W) 1.1 2.2 3.3 4.4 5.5 11 27 54 67.5 81 Protein to DNA Ratio 20 40 60 80 100 200 400 2000 2500 3000 Table VII.3 Reaction conditions for the gel shift assay of SPL and DNA under anaerobic conditions with Apo-SPL. 129 Lane 10 11 Binding Buffer (9L) 50% Gylcerol luL) 94mer oligo (pmol) 0.46 0.46 0.46 0.46 0.46 0.46 0.46 0.46 0.46 0.46 0.46 NP-40 lpL) MQ H20 ltd-1 11 10 SPL (pmol) 2.3 4.6 7.9 9.2 11.5 13.8 16.1 18.4 23 27.6 SPL (nM) 115 230 345 460 575 690 805 920 1150 1 380 Protein to DNA Ratio 10 15 20 25 30 35 40 50 60 AdoMet (nmol) Table VII.4 Reaction conditions for the gel shift assay of SPL and DNA under anaerobic conditions with AdoMet. 130 Samples (Table VII.1 - 4) were prepared in an anaerobic Coy chamber at 4°C except were otherwise noted. Samples were incubated for 2 hours followed by the addition of 2 pL of loading dye. The entire sample was loaded and the gel was run at 200 V for 3 hours. The gel was fixed in 7% acetic acid and then dried between two sheets of cellophane in a drying rack overnight. The gel was cut from the drying racks and visualized by audioradiography (X-OMAT AR-5 film from Kodak). Preparation of Apo-SPL Purified SPL (1 mL, 100 pM) was dialyzed against 2 X 500 mL Buffer A (20 mM sodium phosphate, 500 mM NaCl, 10 mM imidazole, 5% glycerol, pH 8.0) containing 10 mM EDTA for 2 hours at 4 °C followed by dialysis in 2 X 500 mL Buffer A at 4 “C for an additional 1 hour. The protein was removed from the dialysis tubing and concentrated with a Amicon Ultra concentrator with a YM - 10 membrane VII.3 Results and Discussion DNA binding of as isolated protein A 94mer DNA oligomer was prepared based on a sequence from the B. subtilis chromosomal DNA in hopes of Simulating the type of DNA sequences located in endospores. The 94 DNA base sequence was chosen because it is short enough to be easily observed on the polyacrylamide gel after protein binding but long enough to allow multiple proteins to bind one oligo. The protein 131 used in these experiments was isolated as normal and contained ~ 1.9 Fe/SPL; no chemical reductant was added to the protein. The addition of increasing amounts of SPL to the synthesized 94 base pair oligonucleotide shows increasing amounts of the 32F end labeled DNA being retarded in the gel (Figure Vll.1). The dissociation constant was measured by scanning the gel with a GS-900 gel scanner from BioRad and calculating the band density (Table VII.5) of the retarded gel and plotting a linear graph of the [SPL-total] versus v ([SPL-DNA]/[DNA])(Figure Vll.2). One can then calculate the Kd from the point at which v = 0.5 (50% of DNA is bound to protein). The resulting dissociation constant is Kd = 4501100 nM. Lane 1 2 3 4 5 6 7 8 9 10 11 Figure VII.1 Anaerobically isolated SPL binding to a 94 base pair DNA oligomer. An increasing amount of SPL was added ranging from 0 pmol (lane 1) to 46 pmol (lane 11), as detailed in Table Vll.1. As more SPL is added to the DNA oligomer, the DNA band is retarded an increasing amount. A dissociation constant of 450 :l: 100 nM can be calculated by measuring the band densities. 132 The binding constant calculated for SPL falls within a typical range for DNA binding proteins binding non-specifically to DNA.10 For example, DNA photolyase has a KD = 1 uM for non specific DNA binding.1 1 o o 0.8 — :2? O 9 . g. 0.6 - :f' v = O 5 Z 0. d 0.4 - a 1’ o 0.2 — K =4 . / d 50 :l: 100 O l l r r r 0 200 400 600 800 1000 [SPL] nM Figure Vll.2 Plot of [SPL-total] versus v ([SPL—DNA]l[DNA]) for the anaerobically as isolated SPL binding to a 94 base pair DNA oligomer. DNA binding of SPL under aerobic conditions The binding of SPL to our synthesized oligo was measured under aerobic conditions in an attempt to see if the oxidation state or amount of iron affected the DNA binding properties of SPL. It has been noted in SPL that exposure to oxygen results in a loss of protein color and reduced UV spectrum, most likely the result of cluster oxidation and loss of iron from the cluster. 133 The addition of increasing amounts of SPL to the synthesized 94 base pair oligonucleotide shows increasing amounts of the 32F end labeled DNA being retarded (Figure VII.3). The dissociation constant was measured by scanning the gel with a GS-900 gel scanner from BioRad and calculating the band density (Table Vll.5) of the retarded gel and plotting a linear graph of the [SPL-total] versus v ([SPL-DNA]/[DNA])(Figure Vll.4). One can then calculate the Kd from the point at which v = 0.5 (50% of DNA is bound to protein). The resulting dissocitation constant is Kd= 700 t 100 nM. Lane 1 2 3 4 5 6 7 8 9 10 11 Figure VII.3 Anaerobically isolated SPL exposed to oxygen binding to a 94 base pair DNA oligomer. An increasing amount of SPL was added ranging from 0 pmol (lane 1) to 92 pmol (lane 11), as detailed in Table Vll.2. As more SPL is added to the DNA oligomer, the DNA band is retarded an increasing amount. A dissociation constant of 700 :l: 100 nM can be calculated by measuring the band densities. 134 It is therefore observed that the affect of cluster oxidation on the non-specific DNA binding properties of SPL is minimal at best as the increase in K, is almost within experimental error. This gives an early indication that the iron sulfur cluster of SPL has little effect on the DNA binding properties. 1 0.8 _ § 0 z E 2' z o _'i 0. Q 2’ = 700 :I: 100 / K0 0 r T I l r 0 200 400 600 800 1 000 [SPL] nM Figure VII.4 Plot of [SPL-total] versus v ([SPL-DNA]/[DNA]) for the apo-SPL binding to a 94 base pair DNA oligomer. DNA binding of apo-SPL The iron sulfur cluster of SPL was successfully removed by EDTA dialysis and only 0.1 Fe per SPL was found after dialysis. Similar to the above experiment carried out in aerobic conditions, this experiment was designed to determine whether the DNA binding of SPL is affected by the iron sulfur cluster. If 135 the cluster is aiding DNA binding, then we should see a significant affect on the gel by removal of the cluster. Similar to the aS-isolated protein, non-specific DNA binding was seen on the gel-shift assay with an increase in oligo retardation with increasing amounts of SPL (Figure Vll.5); the dissociation constant was measured to be 5501100 nM Figure VII.5 Apo-SPL binding to a 94 base pair DNA oligomer. An increasing amount of SPL was added ranging from 0 nmol (lane 1) to 1.65 nmol (lane 11), as detailed in Table VII.3. As more SPL is added to the DNA oligomer, the DNA band is retarded an increasing amount. A dissociation constant of 550 :l: 100 nM can be calculated by measuring the band densities. The dissociation constant was measured by scanning the gel with a GS-900 gel scanner from Bio-Rad and calculating the band density (Table Vll.5) of the retarded gel and plotting a linear graph of the [SPL-total] versus v ([SPL- 136 DNA]l[DNA])(Figure Vll.6). One can then calculate the Kd from the point at which v = 0.5 (50% cf DNA is bound to protein). As in the experiment carried out under aerobic conditions, there is very little affect on the dissociation constant of SPL and DNA. Removal of the iron sulfur cluster increases the dissociation constant but not by a large amount and is within experimental error. It is, therefore, likely that the iron sulfur cluster is not significantly involved in DNA binding and the cluster binds away from this area of the protein, likely at the C - terminus were SPL shares sequence homology with DNA photolyase (the likely location of a helix-tum-helix DNA binding motif). O 0.8 1 12'" E E; 0-6 ‘ v = 0.5 2 O D. -’ 0.4 - i o ;’ o 0.2 4 . /Kd = 550 1 100 0 k I I I I l 0 200 400 600 800 1000 1200 [SPL] nM Figure VII.6 Plot of [SPL-total] versus v ([SPL-DNA]/[DNA]) for the apo-SPL binding to a 94 base pair DNA oligomer. 137 Based on crystal structures of the other radical SAM superfamily members,”15 the iron sulfur cluster is likely located at the C terminus of an TIM barrel nearer to the N-tenninus of the protein. DNA binding of as isolated protein with SAM The addition of the co-factor AdoMet to SPL has been shown to increase the proteins stability. Since AdoMet is used in the reaction catalyzed by SPL, it is possible that it affects the binding affinity of SPL for DNA. However, since DNA binding likely occurs away from the site of SAM binding at the cluster, it is also possible that non-specific DNA binding may not be affected. SAM was added to the aS-isolated SPL to see if this affects its DNA binding properties. (Figure VII.7) As with the samples without SAM, the more SPL that was added, the more the DNA was retarded on the gel. Calculating the dissociation constant for SPUSAM binding to DNA gives Kd = 5501:100 nM (Figure Vll.8). The dissociation constant was measured by scanning the gel with a GS-900 gel scanner from BioRad and calculating the band density (Table Vll.5) of the retarded gel and plotting a linear graph of the [SPL-total] versus v ([SPL- DNA]/[DNA])(Figure Vll.8). One can then calculate the Kd from the point at which v = 0.5 (50% of DNA is bound to protein). The addition of SAM therefore does not have an effect on the non-specific binding between SPL and DNA, as the dissociation constants are almost identical in the presence and absence of SAM. 138 Lane 1 2 3 4 5 6 7 8 9 10 11 Figure VII.7 Anaerobically isolated SPL with SAM binding to a 94 base pair DNA oligomer. An increasing amount of SPL was added ranging from 0 pmol (lane 1) to 27.6 pmol (lane 11), as detailed in Table VII.4. As more SPL is added to the DNA oligomer, the DNA band is retarded an increasing amount. A dissociation constant of 550 1 100 nM can be calculated by measuring the band densities. VII.4 Conclusions Based on the gel-shift assays carried out throughout this work, SPL can bind non-Specifically to DNA oligomers in the absence of the SP photolesion. It appears that SPL can bind to the DNA in both the presence and absence of the iron sulfur cluster and that the presence of oxygen or SAM makes little difference in the binding capability. This leads one to draw the conclusion that the SPL binds to DNA away from the iron sulfur cluster site. 139 1 ’1 O 0.8 l g 0.6 - <2: v = 0.5 D. d 0.4 - (L) Z O 0.2 - . Kd = 550 :I: 100 0 4 ° . . . . . 0 200 400 600 800 1000 1200 [SPL] nM Vll.8 Plot of [SPL-total] versus v ([SPL-DNA]/[DNA]) for the anaerobically as isolated SPL with SAM binding to 94 base pair DNA oligomer. Lanes 1 2 3 4 5 6 7 8 9 10 11 Anaerobic Figure Vll.1 2.8 5.6 24.7 65 64.3 70.5 80.2 83.4 98.2 83 98 Aerobic 0 5.5 20.4 25.3 37.1 50.2 60.8 70 74.2 80.2 90.4 Figure Vll.2 Apo-SPL 0 9.7 29.5 38.1 52.3 52.3 88.6 96.9 92.6 73.4 66.1 Figure VII.3 SPL w/SAM 0 3.1 12.8 25.9 50.5 67.3 64.9 69.3 91.6 100 100 Figure VII.4 Table VII.5 Percentage of bound protein as calculated from the band density of the gel shift assay. 140 Non-specific DNA binding has been implicated recently as the reason for quick recognition of specific sequences and specific damaged bases by enzymes that interact with DNA.10 The proposed mechanism for an increase in enzymatic activity involves enzyme binding DNA and then Sliding along the DNA helix until it recognizes the specific damage or sequence upon which the enzyme will act.3’ 10 This binding enhances an enzyme’s activity by allowing it to more rapidly recognize substrates along the DNA helix. In recent crystal structures of DNA photolyase, a DNA oligo with a cyclobutane dimer incorporated in the middle was bound to the protein. In this structure, the dimer was observed to flip out of the DNA double helix and into the active site of DNA photolyase.8 This is likely the same method that SPL uses to recognize photoproducts. After the onset of germination, the pH of the cell changes and allows SASP to dissociate from the DNA and get destroyed.16 SPL may then bind loosely to the DNA via a C — terminal helix-tum-helix motif and move up and down the double helix searching for photoproducts that have flipped out of the double helix. Upon recognition of SP, the flipped out bases pair would be centered in the middle of the beta barrel, that is likely the N -— terminal structure of SPL similar to other radical SAM superfamily proteins.12'14 The cluster would than transfer the electron to SAM and cleave the C-S bound to create the 5’-deoxyadenosyl radical that would abstract a hydrogen from the C-6 thymine ring and begin repair. Upon repair, the base pairs would return to their normal configuration in the double helix and SPL would search for other photoproducts to repair. 141 Vll.5 References "1. 10. 11. Sancar, A., Structure and Function of DNA Photolyase and Cryptochrome Blue-Light Photoreceptors. Chemical Reviews 2003, 103, (6), 2203-2237. Suck, D., DNA recognition by DNase I. Journal of Molecular Recognition 1994, 7, (2), 65-70. Woodhead, J. L.; Malcolm, A. D. B., Non-specific binding of restriction endonudease EcoR1 to DNA. Nucleic Acids Research 1980, 8, (2), 389- 402. Lanza, A.; Tomaletti, 8.; Rodolfo, C.; Scanavini, M. C.; M., P. A., Human DNA Topoisomerase l-mediated Cleavages Stimulated by Ultraviolet Light-induced DNA Damage. Journal of Biological Chemistry 1996, 271, (12), 6978-6986. Aravind, L.; Anantharaman, V.; Balaji, S.; Babu, M. M.; lyer, L. M., The many faces of the helix-tum-helix domain: transcription regulation and beyond. FEMS Microbiology Reviews 2005, 29, (2), 231-262. Voet, D.; Voet, J. G., Biochemistry. 2nd ed.; John Wiley & Sons: New York, 1995. Fajardo-Cavazos, P.; Salazar, C.; Nicholson, W. L., Molecular cloning and characterization of the Bacillus subtilis spore photoproduct lyase (spl) gene, which is involved in repair of UV radiation-induced DNA damage during spore germination. Journal of bacteriology 1993, 175, (6), 1735-44. Mees, A.; Klar, T.; Gnau, P.; Hennecke, U.; Eker, A. P. M.; Carell, T.; Essen, L.-O., Crystal structure of a photolyase bound to a CPD-like DNA lesion after in situ repair. Science 2004, 306, (5702), 1789-1793. Slieman, T. A.; Rebeil, R.; Nicholson, W. L., Spore photoproduct (SP) lyase from Bacillus subtilis specifically binds to and cleaves SP (5- thyminyI-5,6-dihydrothymine) but not cyclobutane pyrimidine dimers in UV- irradiated DNA. Journal of Bacteriology 2000, 182, (22), 6412-7. Halford, S. E.; Marko, J. F., How do site-specific DNA-binding proteins find their targets? Nucleic Acids Research 2004, 32, (10), 3040-3052. Lepore, B. W.; Ruzicka, F. J.; Frey, P. A.; Ringe, D., The x-ray crystal structure of lysine-2,3-aminomutase from Clostridium subterrninale. Proceedings of the National Academy of Sciences of the United States of America 2005, 102, (39), 13819-13824. 142 12. 13. 14. 15. 16. Berkovitch, F.; Nicolet, Y.; Wan, J. T.; Jarrett, J. T.; Drennan, C. L., Crystal structure of biotin synthase, an S-adenosylmethionine-dependent radical enzyme. Science 2004, 303, (5654), 76-80. Layer, G.; Kervio, E.; Moriock, G.; Heinz, D. W.; Jahn, D.; Retey, J.; Schubert, W.-D., Structural and functional comparison of HemN to other radical SAM enzymes. Biological Chemistry 2005, 386, (10), 971—980. Layer, G.; Moser, J.; Heinz, D. W.; Jahn, D.; Schubert, W.-D., Crystal structure of coproporphyrinogen Ill oxidase reveals cofactor geometry of Radical SAM enzymes. EMBO Journal 2003, 22, (23), 6214-6224. Haenzelmann, P.; Schindelin, H., Crystal structure of the S- adenosylmethionine-dependent enzyme MoaA and its implications for molybdenum cofactor deficiency in humans. Proceedings of the National Academy of Sciences of the United States of America 2004, 101, (35), 12870—12875. Kosman, J.; Setlow, P., Effects of carboxy-terrninal modifications and pH on binding of a Bacillus subtilis small, acid-soluble spore protein to DNA. Journal of Bacteriology 2003, 185, (20), 6095-6103. 143 CHAPTER VIII INTERACTION OF S-ADENOSYLMETHIONINE WITH THE IRON SULFUR CLUSTER OF SPORE PHOTOPRODUCT LYASE Vlll.1 Introduction A characteristic feature of the radical SAM superfamily is the use of an iron sulfur cluster and AdoMet in activity.1 It has been of significant interest over the years to characterize the interaction between AdoMet and the cluster, with work on PFL-AE, LAM, and BioB giving some of the most detailed results for this interaction and the corresponding adenosyl radical that results. Work on PFL-AE has provided great detail for the interaction between SAM and the iron sulfur cluster. ENDOR and Mdssbauer studies have been able to model this interaction and provide detailed models for SAM binding the iron sulfur cluster via the carboxyl oxygen and amine of SAM with a sulfide-sulfonium interaction possibly providing a pathway for electron transfer.2'5 EXAFS studies on LAM showed binding of SAM to the cluster and an interaction between the sulfonium of SAM and an iron of the cluster.6 This work was followed by ENDOR studies to further characterize this interaction, which provided more evidence for interaction between SAM and the cluster.7 144 To date, there has been little work characterizing the interaction between SPL and AdoMet. In order to probe this interaction, a combination of equilibrium dialysis, ENDOR and Q-band EPR experiments are employed to resolve binding constants, bond coordinations and bond interactions. Equilibrium dialysis has been widely used to obtain dissociation constants for small molecule binding to proteins. This method involves placing a thin membrane with a molecular weight cutoff in between two containers and placing the protein on one side of the membrane and the small molecule on the other side. The small molecule is able diffuse through the membrane and the protein is not; as such, if the small molecule binds to the protein one can measure the difference between the amount of protein on one Side of the membrane versus the other side and calculate how much of the small molecule is bound to the protein. A variety of methods are available to measure the amount of small molecule including UVNis or radiolabeling.8 Our experiment will use radiolabeled SAM to allow for easy quantification of SAM. ENDOR spectroscopy has been applied to a variety of other iron sulfur 9. cluster containing enzymes including aconitase 11 and nitrogenase12 to probe the interaction with substrates. ENDOR spectroscopy examines the electron - nuclear hyperfine interactions which are generally too small to be observed by CW EPR. This can be employed in metalloproteins in which an electron spin (i.e. S = 1/2) such as that of an iron sulfur cluster is in the vicinity of a magnetic nucleus such as 1H, 2H, 13C, 14N, 15N, 17O, 31P or 33$ with l > 0 . Hence, interactions that are too small to be observed with CW EPR can be detected by 145 ENDOR.13 As seen with the PFL-AE and LAM, this technique will allow one observe and model the interaction of SPL’s iron sulfur cluster and isotopically labeled SAM. Vlll.2 Experimental Methods Materials All chemicals used in this work were purchased commercially and were of the highest purity. [2, 8, - 3H] -ATP was purchased from GE Healthcare. Individual Equilibrium Dispo-Dialyzer® 75 uL disposable kits were purchased from the Nest Group Inc. for equilibrium dialysis. lsotopically labeled SAM including 2H, 13C, 15N and 17O SAM had been previously synthesized in our lab?" 4 Synthesis of [2, 8, -3H] SAM SAM labeled with 3H was synthesized by using the following reaction. A 5 mL reaction of 100 mM Tris HCI (pH 8.0), 50 mM KCI, 26 mM MgCl2, 5.2 mM adenosine triphosphate (ATP), 8% B-mercaptoethanol, 1 mM EDTA, 6.8 mM methionine, 2.5 pL Inorganic pyrophosphatase and 500 pL SAM synthetase crude lysate and 1 mCi [2, 8 - 3H] — ATP stirred at room temperature for 16 hrs and quenched with 500 pL 1 M HCI. The reaction was monitored by TLC to completion and purified by loading onto a source 158.6 cationic exchange column. A linear gradient of MO H20 to 1 M HCI was used to elute the [2, 8 - 3H] 146 SAM. The fractions containing SAM were lyophilized and redissolved in 50 mM HEPES, 200 mM NaCl (pH 7.5). Equilibrium dialysis of spare photoproduct lyase Individual Equilibrium Dispo-Dialyzer® 75 uL disposable kits with a molecular weight cutoff of 10,000 Da were used in this experiment. SPL at varying concentrations (75 pM, 125 pM, 175 uM, 250 pM) were place on Side A of the Dispo-Dialyzer® in a buffer containing 20 mM sodium phosphate, 500 mM NaCl, 5% glycerol pH 8.0. Side B of the Dispo-Dialyzer® contained the same buffer and 250 uM [2, 8,-3H] SAM. All work was carried out in an anaerobic mBraun box. These sample were made in triplicate with an additional three samples prepared without protein as a control for SAM diffusion. All samples were placed in a New Brunswick incubator/shaker and left for 2 hours at 100 RPM at 37 °C. Samples were removed from the shaker and the contents of each side of the Dispo-Dialyzer® were pipetted into scintillation vials with 15 mL of liquid scintillation fluid and run on a Beckman LS 6500 liquid scintillation counter. Electron nuclear double resonance spectroscopy and Q-band EPR sample preparation SPL was purified as described in chapter 2 and concentrated to 550 uM. All samples for ENDOR were prepared anaerobically in a mBraun box. 100 uL of purified SPL was reduced with a final concentration 10 mM sodium dithionite and 10 mM DTT on ice for 10 minutes. After reduction, the SAM (either methyl 2H, 147 methyl 13c, amino 15N, carboxyl 17c or non-labeled SAM) was added to the SPL sample and mixed. The sample was then transferred to custom made quartz tube for 35 GHz EPR and immediately frozen with liquid nitrogen. Q-band electron paramagnetic resonance Q-band continuous wave electron paramagnetic resonance was carried out at 35GHz, 2K, 20 dB, and 2.0 G modulation on a modified Varian E-110 Spectrometer with a liquid helium immersion dewar. Electron nuclear double resonance spectroscopy Pulsed ENDOR spectra (35 GHz) were recorded on a Spectrometer described earlier,14 equipped with a helium immersion dewar, and all the measurements were carried out at approximately 2 K. Pulsed ENDOR measurements employed the three-pulse Mims ENDOR sequence (rzl2-r-rzl2-T- 7d2- r-echo), where the RF was applied during the interval T. For a nucleus (N) of spin l = 1/2 (130, 1H,15N) interacting with a S = 1/2 paramagnetic center, the first-order ENDOR spectrum for a single molecular orientation is a doublet, (1) centered at m, the Larrnor frequency, and split by A, the orientation dependent hyperfine constant, when w >A/2, as is true here for 1H, 13C, and 15N nuclei. 148 Similarly, for a deuteron, 2H (i = 1), or 14N (1:1) and 17o (l = 5/2) where m >A/2, as is true here, the first-order ENDOR resonance condition can be written, Vi(i)=VDi—i_ (2) where P, is the orientation-dependent quadrupolar splitting. This is the case with 2H and 170 nuclei in the present study. The full hyperfine tensor of a coupled nucleus, both principal values and orientation parameters (Euler angles) with respect to the g-tensor frame, is obtained by Simulating the 2-D pattern of orientation-dependent ENDOR spectra recorded across the EPR envelope using the procedures and program described elsewhere.""19 For a nucleus with hyperfine coupling, A, the Mims techniques have a response R that depends on the product, Ar, according to the equation. R ~ [1 — cos(27zA 1)] (3) This function has zeroes, corresponding to minima in the ENDOR response (hyperfine suppression holes), at Ar = n; n = 0, 1, 2, and maxima at A1 = (2n + 1)/2; n = 0, 1, 2, ...,15'16 The hyperfine couplings suppressed by the holes at A: n/r, n = 1, 2, 3, can be adjusted by varying 1. However, the central, n = 0, hole at v = m persists regardless. This can be of significance in distinguishing a tensor that is dominated by anisotropic interactions from one that is dominated by isotropic ones. The latter would never predict ENDOR intensity near m, while 149 the former does so for certain orientations. By suppressing intensity near 140, the n = 0. Mims hole diminishes the differences between the two cases. Vlll.3 Results and Discussion Determination of the SAM-SPL dissociation constant Equilibrium dialysis proved successful in obtaining a dissociation constant (Kd) for SPL and SAM. Table Vlll.1 shows the Kd results from experiments run at various concentrations of SAM. Dissociation constants were calculated by using formula (4). Kd = [SAM] * [SPL] l [SPL-SAM] (4) Sample Set 1 2 3 4 [SAM] 250 uM 250 uM 250 uM 250 uM [SPL] 75 “M 125 M 175 M 250 “M Kd-sm 225 pM 175 M 210 pM 185 pM Table Vlll.1 Table of dissociation constants calculated from varying amounts of SPL during equilibrium dialysis. This yielded an average Kd = 200 uM :1: 25 pM. Averaging the results from the different experiments yields a Kd = 200 1M 1: 25 pM. By finding the dissociation constant for the SPL-SAM interaction, it can than be compared to other de of the radical SAM superfamily and provide insight into the mechanism of SPL. Work on biotin synthase showed a low Kd similar to SPL, 150 with Kd= 100 :t 20 pM in the absence of dethiobiotin and K, = 2.3 1: 2 pM in the presence of the substrate dethiobiotin.20 As the substrate for SPL, SP, is not as easy to produce or quantify, no experiments were carried out in the presence of SP lesions. However, if SPL is similar to biotin synthase, an increase in binding affinity should be seen in the presence of it substrate, similar to that of biotin synthase. Synthetically produced SP would allow for the easy manipulation of the equilibrium dialysis experiments and such work is ongoing in our lab. Initial Results from Q-band EPR of reduced SPL Initial results from the Q-band EPR of reduced SPL shows the presence of a [4Fe-4S]1+ cluster similar to that observed for the X-band EPR observed in chapter lV (Figure Vlll.1 and 2). However, the addition of unlabeled SAM dramatically alters the shape of the EPR spectrum. Addition of the labeled SAMs produce spectra that are similar to the reduced SAM but also contains some high spin signals. Initial ENDOR results ENDOR data collection has yet to be completed for ENDOR spectroscopy for samples containing labeled SAM or unlabeled SAM. This work is still in progress and as of yet has revealed no signals due to low coupling between SPL and AdoMet. The absence of the ENDOR signal could be caused by a lack of SAM binding to the iron sulfur cluster or due to a weak signal strength. 151 I T I I 0 5000 Field (G) 10000 15000 Figure Vlll.1 35 GHz CW EPR absorbance spectra of SPL in the absence and presence of SAM A. Reduced SPL. B. Reduced SPL with non-labeled SAM. C-F. Reduced SPL with labeled SAM, (c) 17o SAM; (0.) 15N SAM; (E.) methyl ‘30 SAM; (F.) methyl 2H SAM. 152 O 5000 Field (G) 10000 15000 Figure Vlll.2 35 GHz CW EPR derivative spectra of SPL in the absence and presence of SAM A. Reduced SPL. B. Reduced SPL with non-labeled SAM. C-F. Reduced SPL with labeled SAM, (c.) 17o SAM; (0.) 15N SAM; (5.) methyl ‘30 SAM; (F.) methyl 2H SAM. 153 VIII.4 Conclusions Equilibrium dialysis experiments have shown SPL to have a dissociation constant with SAM of 200 1: 25 (M, which is in the range of that observed for BioB. Further experiments are needed using synthesized SP to see whether binding becomes tighter in the presence of substrate as it does in the case of BioB when dethiobiotin is added.20 The relatively low binding affinity of SPL to SAM could be caused by the lack of substrate or the lack of DNA in the samples or both. Either of these additions might cause tighter binding of SPL to SAM. The expectation is that substrate binding alters the protein confirmation and allows tighter SAM binding. Q-band EPR of SPL shows several interesting phenomenon that are difficult to explain without further experimentation. The observance of the much altered Spectrum of reduced SPL with non-labeled SAM, which is not observed at X-band, is perplexing and could result from sample contamination or an altered SAM binding coordination. No ENDOR signals have been observed to date and work is in progress. 154 Vlll.5 References 1. Cheek, J.; Broderick, J. B., Adenosylmethionine-dependent iron-sulfur enzymes: versatile clusters in a radical new role. Journal of biological inorganic chemistry 2001 , 6, (3), 209-26. Krebs, C.; Broderick, W. E.; Henshaw, T. F.; Broderick, J. B.; Huynh, B. H., Coordination of Adenosylmethionine to a Unique Iron Site of the [4Fe- 48] of Pyruvate Formate-Lyase Activating Enzyme: A Moessbauer Spectroscopic Study. Journal of the American Chemical Society 2002, 124, (6), 912-913. Walsby, C. J.; Hong, W.; Broderick, W. E.; Cheek, J.; Ortillo, D.; Broderick, J. B.; Hoffman, B. M., Electron-Nuclear Double Resonance Spectroscopic Evidence That S-Adenosylmethionine Binds in Contact with the Catalytically Active [4Fe-4Sj+ Cluster of Pyruvate Formate-Lyase Activating Enzyme. Journal of the American Chemical Society 2002, 124, (12), 3143—3151. Walsby, C. J.; Ortillo, D.; Broderick, W. E.; Broderick, J. B.; Hoffman, B. M., An Anchoring Role for FeS Clusters: Chelation of the Amino Acid Moiety of S-Adenosylmethionine to the Unique Iron Site of the [4Fe—48] Cluster of Pyruvate Formate-Lyase Activating Enzyme. Journal of the American Chemical Society 2002, 124, (38), 1 1270-1 1271. Walsby, C. J.; Ortillo, D.; Yang, J.; Nnyepi, M. R.; Broderick, W. E.; Hoffman, B. M.; Broderick, J. B., Spectroscopic Approaches to Elucidating Novel Iron-Sulfur Chemistry in the "Radical-SAM” Protein Superfamily. Inorganic Chemistry 2005, 44, (4), 727-741. Cosper, N. J.; Booker, S. J.; Ruzicka, F.; Frey, P. A.; Scott, R. A., Direct FeS Cluster Involvement in Generation of a Radical in Lysine 2,3- Aminomutase. Biochemistry 2000, 39, (51), 15668-15673. Cosper, M. M.; Cosper, N. J.; Hong, W.; Shokes, J. E.; Broderick, W. E.; Broderick, J. B.; Johnson, M. K.; Scott, R. A., Structural studies of the interaction of S-adenosylmethionine with the [4Fe-48] clusters in biotin synthase and pyruvate formate-lyase activating enzyme. Protein Science 2003, 12, (7), 1573-1577. Voet, D.; Voet, J. G., Biochemistry. 2nd ed.; John Wiley & Sons: New York, 1995. Werst, M. M.; Kennedy, M. C.; Houseman, A. L. P.; Beinert, H.; Hoffman, B. M., Characterization of the iron-sulfur [4Fe-4S]+ cluster at the active site of aconitase by iron-57, sulfur-33, and nitrogen-14 electron nuclear double resonance spectroscopy. Biochemistry 1990, 29, (46), 10533-40. 155 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. Telser, J.; Emptage, M. H.; Merkle, H.; Kennedy, M. C.; Beinert, H.; Hoffman, B. M., Oxygen-17 electron nuclear double resonance characterization of substrate binding to the 4—iron-4-sulfur ([4Fe-48]1+]) cluster of reduced active aconitase. Journal of Biological Chemistry 1986, 261 , (11), 4840-6. Werst, M. M.; Kennedy, M. C.; Beinert, H.; Hoffman, B. M., Oxygen-17, proton, and deuterium electron nuclear double resonance characterization of solvent, substrate, and inhibitor binding to the iron-sulfur [4Fe-4S]+ cluster of aconitase. Biochemistry 1990, 29, (46), 10526-32. Lee, H. l.; Benton, P. M. C.; Laryukhin, M.; Igarashi, R. Y.; Dean, D. R.; Seefeldt, L. C.; Hoffmann, B. M., The interstitial atom of the nitrogenase FeMo-cofactor: ENDOR and ESEEM Show it is not an exchangeable nitrogen. J. Am. Chem. Soc. 2003, 125, 5604-5605. Que, L., Physical Methods in Bioinorganic Chemistry. University Science Books: Sausilito, CA, 2000. Davoust, C. E. D., P. E.; Hoffman, B. M., J. Magne. Reson. 1996, 119, 38. Mims, W. B., In Electron Spin-Echoes. Plenum Press: New York, 1972. Mims, W. B., Proc. R. Soc. London B 1965, 283, 452. Hoffman, B. M.; Venters, R. A., J. Magn. Reson. 1984, 59, 110. Hoffman, B. M.; Martinsen, J., J. Magn. Reson. 1985, 62, 537. Hoffman, B. M., Acc. Chem. Res. 1991, 24, 164. Ugulava, N. B.; Kendra, F. K.; Jarrett, J. T., Control of Adenosylmethionine-Dependent Radical Generation in Biotin Synthase: A Kinetic and Thermodynamic Analysis of Substrate Binding to Active and Inactive Forms of BioB. Biochemistry 2003, 42, (9), 2708-2719. 156 CHAPTER IX GENERAL CONCLUSIONS AND FUTURE DIRECTIONS lX.1 The Iron Sulfur Cluster of SPL Iron sulfur clusters have been found in a multitude of proteins and in nearly all forms of life on the planet." 2 These clusters are involved in a diverse range of chemistry and the type and oxidation state of the cluster found in an enzyme can give important insight into the role the cluster plays in the protein. Previously published work on SPL has shown it to contain an iron sulfur cluster, however, this work was carried out with inactive protein after cluster reconstitution.3 Our work reported herein is the first to examine the as-isolated active iron sulfur cluster of SPL. UVNis spectroscopy of the purified SPL shows a spectrum characteristic of an iron sulfur cluster containing protein, with broad features between 300 and 600 nm, to which extinction coefficients at 410 nm and 450 nm of 11.9 and 10.5 mM'1cm'1 can be assigned. Addition of the reductant, sodium dithionite, causes a decrease in signal characteristic of a redox active iron sulfur cluster. EPR spectroscopy further confirmed the presence of an iron sulfur cluster and was able to give a more complete description of its state. EPR of the as isolated SPL showed a isotropic signal centered at g = 2.02 that is characteristic of a [3Fe- 157 4S]1+ cluster. Spin quantification Shows that this cluster accounted for ~35% of the iron present in the protein. Upon chemical reduction, there is a significant change in the EPR spectrum of SPL, a nearly axial signal is observed with gz = 2.025, gy = 1.928, and 9x = 1.890. This is characteristic of a [4Fe-4S]1+ cluster. Addition of the enzymes cofactor SAM caused no change is the signal’s shape but rather a reduction in intensity. The spin quantification of these spectra showed the [4Fe-4S]1+ cluster to account for approximately of 54% and 11%, respectively of the iron present in the protein. Mbssbauer spectroscopy was also employed to look at the cluster states as EPR can only look at those states that have an unpaired electron. Mdssbauer of the as isolated cluster yielded a result that 53% was in the [2Fe-28]2+ state and the rest is in the [3Fe-4S]1+ state. Mdssbauer of the chemically reduced SPL like the EPR spectrum, is dramatically altered and shows a mixture of 90% [4Fe- 4S]1+ and 10% [4Fe-4S]2+. The large isomer shift observed in the spectrum of the reduced SPL indicates an electron rich environment. This electron rich environment would facilitate electron transfer from cluster to the C-S bond of SAM. This supports the proposal that SPL and the radical SAM superfamily in general use a [4Fe-4S]1+ cluster to deliver an electron to AdoMet, cleaving the C- S bond to form a putative adenosyl radical and methionine. 158 lX.2 Catalytic activity of SPL Previous reports of SPL did not provide a specific activity of the enzyme?"6 and other reports of SPL activity with SP not incorporated into a DNA helix,7 Show a somewhat low activity. In this report, we provide a specific activity of SPL with the SP lesion in a DNA backbone of plasmid DNA and correlate this activity to the amount of [4Fe-4S]1+ cluster present. Our initial time course assays for SPL provide an activity of 0.33 pmol SP repaired/min/mg SPL. Although earlier publications do not specify a specific activity for SPL, by looking at the results we were able to calculate a specific activity for some of these experiment and it is almost 1000 fold lower than what we have calculated here, possibly because of low Fe—S cluster content and aerobic conditions in their work.6 They also carried out their assay overnight, which may have been longer than necessary to repair the DNA. Despite the high activity of SPL Shown above, EPR studies have shown that it is very difficult to reduce the cluster 100% even with a substantial amount of chemical reductant added. Therefore, we reduced SPL prior to use in the assay and monitored the reduction by EPR. This protein was then used in repair assays. Not surprisingly, by reducing the protein before use in repair assays, an increase in activity to 1.33 uMol SP repaired/min/mg SPL was seen and 2.4 pmol SP repaired/min/mg when corrected for the amount of [4Fe-4S]1+ cluster. There are several implications of these observations. With the enzyme showing an increase in activity corresponding to an increase in the amount of reduced 159 cluster, it is reasonable to conclude that the [4Fe-4S]1+ cluster is the active state of SPL. This conclusion is supported by the evidence for other members of the radical SAM superfamily using the [4Fe-4S]1+ cluster as the active cluster form.8’ 9 It also consistent with a mechanism in which an electron from the electron rich iron sulfur cluster is transferred to the electron deficient sulfonium atom of AdoMet to cause homolytic cleavage of the C-S bond to form the putative adenosyl radical and initiate radical catalysis. IX.3 Mechanism of SPL While many of the enzymes in the radical SAM superfamily utilize AdoMet as a substrate including biotin synthase10 and pyruvate formate lyase activating enzymes, another well characterized enzyme, lysine 2,3-aminomutase", utilizes SAM as a catalytic cofactor. Mehl and Begley first published in 1999 a mechanism in which SPL was used as a catalytic cofactor.12 This mechanism was later supported by evidence from Cheek and Broderick showing that tritium transfers from SP C-6 to SAM during SP repair.13 The mechanism proposed in Scheme Vl.1 also predicts that a hydrogen atom from a 5’-deoxyadenosine intermediate is abstracted by the product thymine radical (Scheme Vl.1). If correct, this predicts that the label from 5’- tritiated SAM should be transferred to repaired thymine. To support this, we synthesized AdoMet that was labeled at the 5’- position and looked for incorporation into thymine after SP repair. A peak was observed at the location of 160 thymine after HPLC separation of the repair reaction; the quantity of label transfer was consistent with an isotope affect of ~15.8, based on the specific activity of SPL and the amount of tritium incorporated into the SAM. This transfer from SAM to thymine during repair further supports the mechanism proposed. Based on the mechanism originally proposed by Mehl and Begley, AdoMet acts as a cofactor, if this is true, no SAM should be cleaved irreversibly to 5’-deoxyadenosine and methionine. Studies in this report show that after multiple turnover and SP repair, only a small fraction of SAM is cleaved even after 24 hours. This evidence of essentially no irreversible SAM cleavage in addition to other studies in our lab that Show a single SAM can repair upwards of 400 SP lesions13’ 14 strongly supports a repair mechanism in which SAM is utilized as a cofactor. Other mechanistic studies described in this work examined the isotope effect of initial H atom abstraction from the C-6 ring of thymine. By labeling the SP at C-6 position and looking at the amount of tritium transfer at 0%, 25%, 50% and 100% repair we observe an isotope affect of ~15-17, showing that initial H atom abstraction to be a slow step in the reaction pathway. Furthermore, we have calculated the amount of label transfer expected from our labeled C-6 SP to SAM if 100% of the SP is repaired and 100% of the tritium is transferred from SP to SAM. We observe that only 50% of the total amount of label is transferred from the C-6 labeled SP to SAM. One of the predictions that could be made from this result is that SP is formed in a non-stereospecific manner. If this is the case, then the C-6 tritium label will have a stereochemistry that is 50% R and 50% S. It then 161 follows that SPL stereospecifically abstracts the tritium from either 100% of the R isomer or 100% of the S isomer, leaving 50% of the label in the repaired thymine and 50% of the label gets transferred to SAM. In this scenario, SP is formed non- stereospecifically, and SPL repairs SP stereospecifically. Our overall conclusion is that experimental evidence in our lab supports the original mechanism proposed by Mehl and Begley12 in which the H atom is abstracted from the C—6 of thymine stereospecifically by the 5’-deoxyadenosyl radical created by reductive cleavage of AdoMet. The subsequent C-6 radical undergoes a radical mediated B-Scission, causing the C-C bond to break and leaving a radical on the methyl group. This radical re-abstracts a hydrogen from the 5’-deoxyadenosine to reform the 5’-deoxyadenosyl radical which in turn can interact with methionine to reform SAM. IX.4 Interaction of DNA and SPL Most proteins that act upon DNA, either as promoters, inhibitors, replicators or repairers first bind to the DNA and than find the specific sequence or base pair(s) where they will perform their task.15 Often times this non-specific interaction is what allows the enzyme to quickly act upon the DNA base pairs of interest.15 SPL has been shown to be no different and reports herein have shown it capable of binding to DNA in the absence of spore photoproduct with or without the enzymes cofactors, SAM and an iron sulfur cluster. As SPL has C-terminal homology to the repair enzyme DNA photolyase and amino acid residues consistent with a helix-tum-helix DNA binding motif 162 similar to DNA photolyase”, it is likely that SPL binds non-specifically to the DNA via this motif. Studies with DNA photolyase have shown the protein binding to DNA via the helix-tum-helix motif of the C-terrninus with the damaged DNA bases flipped out of the DNA double helix and into the protein’s active site." This is likely the same mechanism by which SPL recognizes the damaged lesion as well, with the thymine bases flipped out of the helix and into the active site near SAM. IX.5 An Overview of Spore Photoproduct Repair Evidence provided above and similarities to other DNA repair enzymes and enzymes in the radical SAM superfamily can provide us with a hypothesis for a complete picture of SPL-catalyzed repair of the spore photoproduct lesion. After the sporulation and production of the SASPS, which binds to the chromosomal DNA with a high affinity under spore like conditions (i.e. pH ~6.5 with dipicolinic acid and Ca2+), the DNA converts to an A-like conformationm‘21 Exposure to ultra-violet light, while in the spore form, causes the DNA lesion spore photoproduct to form, possibly because of the conformation change provided by SASPS binding the DNA.22’ 23 When conditions favor spore germination, the pH of the spore changes back to ~75 and the SASP is degraded24, causing it to unbind the DNA, which changes the conformation back to B. SPL can then non-specifically bind to the DNA via .a helix-tum-helix motif at the C-temlinus and begin searching for SP 16,17 lesions. Similar to cyclobutane dimers and 6’-4’ photoproducts , it is likely 163 that the damaged dimer is flipped out of the DNA helix where SPL can recognize and bind to the SP lesion. The SP dimer is positioned close to the active site of SPL near SAM and the [4Fe-4S]1+ cluster. Once in position, the Fe-S cluster donates an electron to the C-S bond of AdoMet and causes homolytic SAM cleavage, creating a 5’-deoxyadenosyl radical. The newly formed radical Species abstracts a hydrogen from the C-6 ring position of thymine, which than attacks the methylene bridge of the SP lesion. The methylene bridge is cleaved and a radical is formed on the methyl group. This radical reabstracts a hydrogen from 5’-deoxyadenosine to reform the adenosyl radical and can then interact with methionine to remake AdoMet. This is illustrated in Scheme Vl.1. With the lesion repaired, SPL can move along the helix again to repair more SP lesions. IX.6 Implications For DNA Repair And Similarities To DNA Photolyase In addition to being consistent with our current understanding of SPL, the mechanistic proposal from the previous section (IX.5) displays intriguing parallels to the proposed mechanism for DNA photolyase. In both cases, radical chemistry is used to cleave a covalent pyrimidine dimer, with the DNA photolyase using radical anion chemistry and SPL using neutral radical chemistry. These two enzymes, however, use very different mechanisms by which to initiate the radical chemistry as follows: NADH, a secondary cofactor, and light in the case of DNA photolyase, and an iron sulfur cluster and AdoMet in the case of SPL. Thus, radical chemistry is not only a significant source of DNA damage but is also an 164 important means of repairing DNA damage in vivo. It is intriguing that such vastly different cofactors are utilized to perform what is quite similar chemistry. By analogy to what Beinert25 has proposed for other enzyme systems, perhaps the F-S/AdoMet cofactor combination found in SPL is a primitive “holdover” from more anaerobic times, and the NADH and light-driven reaction is a more modern adaptation. lX.7 Further Experiments: Protein Crystallography A fundamental tool when examining enzymes and proteins is x-ray crystallography. By first crystallizing a protein out of solution and than exposing it to x-rays and viewing their diffraction through the crystal, a pattern emerges that can be solved to obtain the three dimensional structure of the protein. This technique has been widely applied to both organic and inorganic materials as well as proteins to solve their three dimensional atomic structures. X-ray crystallography has been responsible for the majority of known protein structures.26 Crystallization of macro-molecules such as proteins is not a trivial manner and can require extensive manipulation of buffering conditions and different crystallization techniques. For the crystallization to be successful, the protein in question must be free of contaminants and in a homogeneous state. After crystallization, the crystals produced must generally be of a resolution of 4 A or better to allow for the successful solution of the 3-D structure.26 165 Work on the protein crystallization of SPL is currently under way. Samples of purified SPL were prepared with an iron content of 3.0 Fe/protein and 5 mM AdoMet as stated in Chapter II followed by concentration to 25 mg/mL. These samples are currently undergoing crystallization studies with our collaborators in the Drennan lab at MIT. Currently, no suitable crystals have been obtained although some poor crystals of SPL have resulted but can not be used for X-ray diffraction. If successful, the crystal structure would confirm the probe of an interaction between SAM and the iron sulfur cluster as well as examine the presence of the helix-tum-helix binding motif. lX.8 Further Experiments: Synthesis of Synthetic Spore Photoproduct It is both difficult and time consuming to create the tritiated SP in the pUC18 plasmid that is used for repair assays in this work. In addition it is difficult to control the amount of SP produced and used in each activity assay. It is therefore very desirable to produce a synthetic spore photoproduct. Work on the synthesis of Spore photoproduct was initially carried out in the Begley lab”, however, these experiments did not provide any evidence for repair activity using SPL. More recent studies have Shown repair of a synthesized SP lesion but the activity was relatively low and SPL could only repair one of the two isomers produced.28 Neither of these reports incorporated a phosphate group in between the dimers or incorporation of SP into a DNA strand, making them far from ideal substrate models for SP. New reports have shown SPL to repair the 166 synthetically produced SP with a phosphate linker but still have not incorporated it into a DNA strand to produce an ideal model of the SP lesion.29 Work is, therefore, in progress in our lab to synthesize an SP lesion and incorporate it into a DNA strand and to use this for further studies of SPL, including kinetic and mechanistic assays as well as spectroscopic studies. A synthetic SP lesion could also be used in coordination with X-ray crystallization studies to obtain a structure with a bound DNA substrate similar to what was obtained with DNA photolyase and a cyclobutane dimer.17 lX.9 Further Experiments: EXAFS studies of SPL Extended X-ray absorption fine structure spectroscopy has been carried out on several members of the radical SAM superfamily, including LAM3O' 31, PFL-AE32 and biotin synthase”. In the studies with LAM, EXAFS has shown and interaction between the Fe of the cluster and selenium used in the place of sulfur in AdoMet.31 However, work on both PFL-AE and biotin synthase, did not show this interaction32, leading to the possibility that members of the radical SAM superfamily that use AdoMet as a catalytic cofactor interact with AdoMet differently than those that use it as a substrate. It would therefore be interesting to apply EXAFS to SPL and see if the interaction between the selenium and Fe exists as it does for LAM and if this correlates to AdoMet’s role as a catalytic cofactor in these enzymes. Currently, we have synthesized a suitable amount of seleno-SAM by using seleno- 167 methionine in our synthesis reaction in the place of methionine and have produced ~20 mg for use. However, EXAFS have not been carried out because of the difficulty in obtaining a sufficiently high concentration of SPL for the experiment. It is expected that a concentration of at least 1 mM SPL would be needed to observe the interaction with EXAFS and this has not been obtained. IX.1O Further Experiments: DNA Binding Studies Past studies have shown SPL to bind to the SP lesion by DNA footprinting6 however this work was carried out with protein that was either reconstituted or did not contain a stoichiometric amount of iron. 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