Construction, Properties and Crystal Structures of Site—Directed Mutants of Tyrosyl—tRNA Synthetase by Katherine Anne Brown A Thesis presented in partial fulfilment of the requirements for the Degree of Doctor of Philosophy of the University of London Biophysics Section Blackett Laboratory Imperial College London 1 ABSTRACT Recent work in the relatively new field of protein engineering has consistently shown that one can alter the functional properties of a protein by selectively mutating a single amino acid. The aim of this research is to discover the physical origins of the change in properties. Residue threonine—51 of tyrosyl—tRNA synthetase from Bacillus stearothermophilus had been selectively changed to a proline (mutant Pro51) using site-directed mutagenesis techniques. The resulting enzyme demonstrated a marked increase in its affinity for tyrosyl adenylate, a stable intermediate in its reaction pathway. Kinetic studies of a double mutant containing the Pro51 mutation and a second mutation (histidine 48 to a glycine; HG48) suggested that the effect of the Thr51-»Pro change might be mediated through a structural change involving the peptide backbone. The crystal structure of the Pro51 mutant was determined and did not reveal any mainchain movement. It emphasized the importance of other seemingly minor effects of this mutation; particularly changes in local hydrophobicity and possibly, configurational entropy of this region of the active site. Crystal structures of three other mutations at position 51 were also determined and served to correlate general trends of catalytic activity with structural data. This information was used to design two mutants of the TyrTS which were constructed in an attempt to define some of these postulated physical effects. Kinetic data provided an indication of the success of this experimental approach. The crystal structure of the HG48 mutation was also determined in order to examine possible structural changes which might contribute to the non —additive kinetic effects when the Pro51 mutation is present. The appearance of a well-ordered water molecule which replaces the missing imidazole ring of the histidine in this mutant provides evidence for a proposed mechanism for the hydrolytic degradation of enzyme—bound tyrosyl adenylate. 2 ACKNOWLEDGEMENTS The work in this thesis represents a collaborative effort between the Biophysics section of the Physics Department and the Biological Chemistry Division of the Chemistry Department both at Imperial College and I should like to thank all members of both groups who have helped me with various aspects of my work. This list would be too numerous to mention by name in the space here, however, certain members have given me particular attention and I should like to specifically acknowledge their contributions to this work. I must first thank my supervisor, Professor David M. Blow, F.R.S., for his advice, encouragement, enthusiasm and unwavering belief in the success of the project and my ability to see it through. I should also like to thank Professor Alan R. Fersht, F.R.S. for providing me with the facilities and necessary expertise to carry out the genetic engineering and enzymological studies presented in this thesis. I am very grateful for all the technical assistance I received in both laboratories and in this respect I thank John Akins, Lesley Lloyd and Geraldine Davies for their help. In the Biophysics section of Imperial College I wish to thank Dr. Peter Brick and Dr. Patrice de Meester who have given me valuable advice and assistance. In the Chemistry Department special thanks are due to Jack Knill—Jones, Dr. Tim Wells, Dr. Robin Leatherbarrow, Dr. Thor Borgford and Tammy Gray who helped me with the genetic engineering and kinetic studies presented here. For their help in the preparation of this thesis I am indebted to: Dr. Peter Brick and Dr. Tim Wells for helpful discussions and proof-reading of this thesis; Dr. Patrice de Meester and Dr. Kim Hendrick for help with preparation of the diagrams; Nick Jackson for photography; and Bryan Driscoll who has become my P.A.. Finally I'd like to thank my family and friends in the U.S. and the U.K. for their support and encouragement through the last three years and a half years. 3 CONTENTS Abstract 1 Acknowledgement 2 Contents 3 List of Figures 7 List of Tables 9 Abbreviations 10 Chapter 1. Introduction 1.1 Protein Engineering: Structure as an Integral Part 11 1.2 Case Studies 12 1.2.1 Introduction 12 1.2.2 Stability 12 A. Destabilization of the Folded State: T4 Phage Lysozyme 13 B. Enhancing Stability: Disulfide Bonds 15 C. Enhancing Stability: Alternative Approaches 17 D. Protein Folding: Proline Isomerization 18 1.2.3 Ligand Binding and Catalysis 19 A. Ligand Binding 20 B. Identification of Active Sites: Domain Structures 23 C. Probing the Energetics of Enzyme Catalysis 25 D. Specificity 27 1.2.4 The Conformation of Polypeptides: Structural Elucidation 29 1.3 Project Aims 30 Chapter 2. Oligodeoxynucleotide — Directed Mutagenesis 2.1 The Automated Synthesis of Oligonucleotides 33 2.1.1 Introduction 33 2.1.2 Materials 34 2.1.3 Method 35 2.2 The FPLC Purification of Oligonucleotides 36 2.2.1 Introduction 36 2.2.2 Materials 36 2.2.3 Method 37 2.3 Site—Directed Mutagenesis 37 2.3.1 Introduction 37 2.3.2 Materials 40 A. Mutagenesis Using the Single Priming and the 40 Amber Selection Approaches 4 B. Mutagenesis Using the Kunkel Method 43 2.3.3 Methods 44 A. Small Scale Preparation of Single—Stranded Ml 3 44 Template for the Single Primer and Amber Selection Approaches B. Large Scale Preparation of Single—Stranded Ml3 45 Template for the Kunkel Method C. Kinasing Mutagenic and Selection Oligonucleotides 46 D. Annealing, Extension, and Ligation 46 E. Preparation and Transformation of Competent Cells 47 F. Phage Supernatant Screening by Hybridization with 47 Mutagenic Oligonucleotides G. Plaque Purification 50 2.4 Dideoxy Sequencing of the tyrS gene 50 2.4.1 Introduction 50 2.4.2 Materials 51 2.4.3 Method 52 2.4.4 Summary of Sequencing Primers 53 2.5 Results 53 Chapter 3. Purification and Kinetic Analysis 3.1 Ami noacyl — tRN A Synthetases 58 3.2 The Expression and Purification of Tyrosyl —tRNA Synthetase 58 3.2.1 Introduction 58 3.2.2 Method 59 A. Preparation of Phage Cultures 59 B. Verification of Overexpression 60 C. SDS Gels 60 D. Infection of Cultures for Preparative Yield of Protein 62 E. Cell Harvesting and Breakage 62 F. Heat Treatment and Ammonium Sulfate Fractionation 62 G. DEAE—Sephacel Ion Exchange Chromatography 63 H. Fast Protein Liquid Chromatography 63 I. Hydroxyapatite Column Chromatography 64 J. Sample Purification 65 3.3 Kinetic Characterization of TyrTS 65 3.4 Active Site Titration 69 3.4.1 Introduction 69 3.4.2 Method 70 5 3.5 Pyrophosphate Exchange Kinetics 70 3.5.1 Introduction 70 3.5.2 Method 71 3.5.3 Results 71 3.6 Pre—Steady State Formation and Pyrophosphorolysis of 73 Enzyme —Bound Tyrosyl Adenylate Complex 3.6.1 Introduction 73 3.6.2 Method 73 3.6.3 Results 75 3.7 The Reaction Profile 79 3.7.1 Calculation of Energy Levels 79 3.7.2 Discussion g0 3.7.3 Uncertainties Due to Temperature Variation g2 Chapter 4. Crystal Structure Determination and Refinement 4.1 Structural Studies of the Tyrosyl —tRNA Synthetase 33 4.1.1 Structure of the Wild —Type TyrTS 33 4.1.2 The Active Site 35 4.1.3 Truncated Mutant 35 4.1.4 Model Building of the Transition State 37 4.2 Crystallization of TyrTS 39 4.2.1 Method 39 4.2.2 Summary of Mutants Crystallized 91 4.3 Data Collection 93 4.3.1 X—ray Sources 93 4.3.2 Photographic Methods 94 4.3.3 The Arndt —Wonacott Oscillation Camera 94 4.4 Data Measurement 96 4.4.1 Densitometry 96 4.4.2 Data Measurement Programs 96 4.4.3 Data Scaling Programs 97 4.4.4 Data Collection and Processing Statistics 99 4.5 Difference Electron Density Map Calculation 99 4.5.1 Theory 99 4.5.2 Programs 101 4.5.3 Results 103 4.6 Refinement 108 4.6.1 Hendrickson — Konnert Restrained Least—Squares 108 Refinement 4.6.2 Method 109 6 4.6.3 Results 109 4.7 Discussion 121 Chapter 5. Discussion 5.1 The Thr51-»Pro Mutation 126 5.1.1 Introduction 126 5.1.2 Kinetic Characterization 127 5.1.3 The Double Mutant Test 130 5.1.4 The Crystal Structure 133 5.1.5 Discussion 136 5.2 The Position 51 Series 138 5.2.1 Kinetic Characterization 138 5.2.2 Linear Free Energy Relationships 140 5.2.3 Crystal Structures 142 5.2.4 Solvent Accessibility Calculations 146 5.2.5 Discussion 150 5.3 The Thr51-»Val Mutation: Testing 'Hydrophobicity' 151 5.3.1 Design of the Mutation 151 5.3.2 Kinetic Characterization 152 5.3.3 Discussion 153 5.4 The Ala50-»Pro Mutation: Prolines in Helices 156 5.4.1 Design of the Mutation 156 5.4.2 Discussion 161 5.5 The His48-*Gly Mutation 163 5.5.1 Kinetic Characterization 163 5.5.2 The Hydrolysis of Tyrosyl Adenylate 163 5.5.3 The Crystal Structure 166 5.5.4 Discussion 171 5.6 Concluding Remarks 174 Appendices I. Materials 176 II. Refined Coordinates and Temperature Factors 178 of New Atoms in Mutant Crystal Structures III. Coordinates Shifts and Temperature Factor 181 Differences References 194 7 LIST of FIGURES 2.1 Elution profile for FPLC purification of an oligonucleotide 3g 2.2 Phage supernatant screening by hybridization with a 49 mutagenic oligonucleotide probe 2.3 Dideoxy sequencing to verify the presence of the Thr51 — >Val 54 mutation 3.1 Overexpression of the Thr51 —> Val TyrTS mutant 61 3.2 DEAE—Sephacel purification of the Thr51 — >Ser TyrTS mutant 67 3.3 Hydroxyapatite purification of the ThrSl—>Ser TyrTS mutant 68 3.4 Pyrophosphate exchange data for the TyrTS TV51 mutant