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 72 3.5 Tyrosyl adenylate formation data for the TyrTS TV51 mutant 76 3.6 Pyrophosphorolysis data for the TyrTS TV51 mutant 77 3.7 Gibbs' free energies of enzyme —bound complexes gl 4.1 The crystal structure of tyrosyl —tRNA synthetase g4 4.2 The active site of TyrTS with bound tyrosyl adenylate g6 4.3 The tyrosine substrate in the binding pocket gg 4.4 The pentacoordinate transition state in the active site 90 of TyrTS 4.5 Construction of the isomorphous difference approximation 102 for the acentric case 4.6 The wild—type difference electron density maps of 106 TyrTS mutants superimposed on the wild —type structure 4.7 The refined structures of wild —type and mutant TyrTS's 112 4.8 Scatchard plot of two non —identical binding sites 124 5.1 Gibbs' free energies of enzyme —bound complexes of position 129 51 mutants 5.2 The energy of interaction of each side chain with the 132 transition state(kcal/mol) in the amino acid activation reaction 5.3 The Ramachandran plot of residues 45 — 60 of the 134 wild—type TyrTS structure 5.4 Hydrogen bonding in the wild —type and TP51 mutant TyrTS 135 structures 5.5 Linear free energy relationships for TyrTS reactions 141 5.6 Changes in binding energy for the formation of tyrosyl 14la adenylate 5.7 Slices of van der Waals* envelopes fo the wild —type TyrTS i4g and TP51 mutant structures 8
5.8 Comparison of the modelled TV51 side chain with other 154 TyrTS structures 5.9 Energy contour diagrams for an L-alanyl residue 15g 5.10 Gibbs' free energies of enzyme —bound complexes of the 155 HG48 mutant 5.11 The linear free energy relationship between the tyrosyl 157 adenylate hydrolysis rate constant and dissociation equilibrium constant 5.12 Van der Baals' radii of the entrance to the active site 170 of the TyrTS 5.13 Model for the hydrolysis of the tyrosyl adenylate in the 172 HG48 and HA48 mutants 5.14 Van derWaals. radii of the active site of TyrTS 173 9
LIST of TABLES
2.1 Primers used for mutagenesis of the tyrS gene 42 2.2 Summary of bacterial strains 42 2.3 Primers used for sequencing the tyrS gene 55 2.4 Summary of results 56 3.1 Purification scheme of mutant TyrTS's 66 3.2 Summary of rate and dissociation constants 78 3.3 Gibbs' free energies of complexes of the wild —type and 78 TV51 TyrTS enzymes 4.1 Crystallization summary 92 4.2 Summary of data collection 100 4.3 Deviations from ideal geometry after and final 111 R—factors 4.4 R.M.S. differences in A between the atomic positions of 116 residues 45—55 (mainchain and side chain) of wild —type TyrTS and each mutant 4.5 Temperature factors of mainchain atoms of residues 45 117 to 55 in wild—type and mutant TyrTS's 5.1 Pyrophosphate exchange activity of TyrTS's 128 5.2 Aminoacylation activity and the Gibbs' free energy of 128 the enz yme — tyrosyl adenylate complex of TyrTS's 5.3 Contributions of individual groups at position 51 to 137 binding energy of ATP in the transition state 5.4 Gibbs' free energies of complexes of wild —type and 139 mutant TyrTS enzymes 5.5 Difference in Gibbs' free energy between bound complexes 139 of each mutant TyrTS and the wild —type enzyme 5.6 Surface accessibilities of wild —type TyrTS and position 147 51 mutants 5.7 The tp,\J/ angles of residues 45 — 55 in the wild —type TyrTS 159 and TP51 mutant crystal structures 5.8 Contact distances for proline in the TP51 and AP50 162 mutants 5.9 Pyrophosphate exchange activity of wild —type and mutant 164 (position 48) TyrTS's ABBREVIATIONS
0 A Angstrom unit - 10"^®m A deoxyadeny1 a te , absorbance a , b, c unit cell edges AMP, ATP adenosine 5 '-monophosphate, adenosine 5 '-triphosphate B temperature factor - 8 t2u , where G - mean displacement of atoms C, CTP deoxycytidylate, cytidine 5 '-triphosphate cal calculated (as a subscript) DEAE diethylaminoethyl DTE di thioerythri tol E enzyme, tyrosyl-tRNA synthetase F structure factor G, GTP deoxyguanylate, guanosine 5 '-triphosphate h,k, 1 Miller indices Inh inhibitor, tyrosinyl adenylate 2-ME 2-me rcaptoe t hano1 obs observed (as a subscript) P phosphate PMSF phenylmethanesulphonyl fluoride PPi pyrophosphate SDS sodium dodecyl sulphate T, dTTP deoxythymidylate, deoxythymidine 5 '-triphosphate Tr i s t r i s (hydroxyme t hy1am inome t hane Tyr ty ro sin e Tyr-AMP tyrosyl adenylate tyrS, TyrTS tyrosyl-tRNA synthetase U, UTP uracil, uridine 5 '-triphosphate YRSI tyrosyl-tRNA synthetase containing tyrosinyl adenylate
Mutation from Xxx to residue Yyy can be abbreviated to XY* where * - the residue number 11
CHAPTER 1
INTRODUCTION
1.1 Protein Engineering: Structure as an Integral Part
As Perutz confronted his model, I began to perceive that knowing all he did about the structure o f hemoglobin he was still baffled that the struc ture failed to explain how the molecule functioned. The structure was in tricate, the bafflement elementary. Why was the hemoglobin molecule so large, so complex, so irregularly but specifically folded up, when all that it had to do was load and unload four oxygen molecules each made o f two atoms? By what mechanism did the structure change as oxygen came on or off? And to what effect?
from The Eighth Day of Creation (1979)
Horace Freeland Judson
Knowledge about the functional characteristics and the three-dimensional structure of
a protein molecule is not necessarily a guarantee for understanding the relationship
between the two. To achieve such an understanding often relies upon designing
experiments which can only be carried out through a collaborative effort between fields of
research which specialize in either functional or structural studies. For example, the
identification of an active site in an enzyme may rely upon the work of a chemist who can provide a substrate analogue which can be retained in a protein crystal suitable for diffraction studies. A crystallographer's structure may then reveal information about which residues are responsible for catalytic activity. However, a study such as this often provides little information about the mechanistic significance of individual residues. Covalent modification of both substrates and residues may provide some information regarding the contribution of an isolated residue to a binding or catalytic process as could studies on natural variants of a particular protein. With the advent of site—directed mutagenesis, though, the scientific community was provided with one of the most invasive means of examining structure—function relationships. Specifically, a genetic engineer/molecular biologist now has the ability to alter the sequence of a cloned gene which could 12 potentially code for a protein with any amino acid substituted at any position. A protein
chemist can then study the physical properties of such a mutant protein while a
crystallographer can examine how such alterations affect structure. The exploitation of
site—directed mutagenesis in the context of such functional and structural studies has
generated the relatively new field of protein engineering.
The generally accepted prerequisites for a protein engineering study are: a cloned
gene; an expressed and purified protein; and a high resolution structure. Admittedly, some
of the original protein engineering studies which examined properties of protein transport
and secretion (Charles et al., 1982; Inouye et al., 1982) were not greatly hindered in their
interpretation by lack of high resolution structural information. However, in the early
studies of Q—lactamase which examined the role of catalytic residues (Dalbadie—McFarland
et al., 1982; Sigal et al., 1982), the functional significance of mutations could not be
adequately interpreted because the structural organization of the active site was not known.
As the field of protein engineering developed it became more apparent that without a
model structure from which to base designs and test theories, full exploitation of many
projects could not be achieved (for detailed reviews see Leatherbarrow and Fersht, 1986;
Knowles, 1987; and Shaw, 1987).
1.2 Case Studies
1.2.1 Introduction
This section is not intended to be a general review of projects in the field of protein engineering to date but rather aims to describe some of the specific contributions of structural studies. This chapter concludes by outlining, in a similar context, the aims of the studies detailed in this thesis.
1.2.2 Stability
Understanding the interactions that govern the folding and subsequently, the thermodynamic stability of proteins has become one of the major areas of interest addressed by protein engineering. According to Matthews (1987), the problem is 13
'complicated not only by the size and complexity of macromolecules but also by the necessity to account for very small energy changes in both the folded and unfolded forms.'
Admittedly, analyses of mutant proteins have provided few guiding principles regarding the
chemical nature and location of amino acid substitutions that alter protein stability
(reviewed by Matthews, 1987). In general, the effects of any given mutation appear to be
dependent on the nature of the substitution as well as the enviroment in which it occurs.
This can also be complicated by the fact that a further segregation of features occurs
between mutants that decrease or enhance such thermodynamic stabilization. In either case
though, the need for three-dimensional structural knowledge is essential for both the
design and interpretation of most designed amino acid alterations. Furthermore, a true
comparison of the functional differences which are directly related to a change in stability
would be incomplete without a direct comparison of the protein structures under study.
1.2.2A Destabilization of the Folded State: T4 Phage Lysozyme
The temperature—sensitive mutants of bacteriophage T4 phage lysozyme comprise a special set of proteins which have been produced initially by random chemical mutagenesis in combination with a thermally selective genetic screen (Streisenger et al., 1961). In this instance, positions in the protein which were known to cause thermal instability served later as targets for site—directed mutagenic studies. However, the main strength in the interpretation of the results from all studies has been the ability to examine the structural alterations of both types of mutations using X—ray crystallographic techniques.
The structure of the Arg69-»His mutant of the T4 phage lysozyme, produced by random mutagenesis, permitted the first direct comparison of two protein structures in which all differences were directly related to a change in thermal stability (Grutter et al.,
1979). Except for the replacement of a partially exposed arginine by a histidine, the refined structure of the isomorphous mutant crystals provided no evidence for structural changes greater than a few tenths of an angstrom. The primary explanation for the observed thermal instability was a presumed destabilization of a hydrophobic core structure by the hydrophillic nature of the new histidine residue. In contrast, the crystal structure of the Glyl 56-»Asp mutant (Gray and Matthews, 1987) demonstrated a forced movement of 14 approximately 0.6X of a portion of the peptide backbone with shifts which propagated up to 10A across the surface of the protein molecule. Without such movement the larger aspartate residue at position 156 would have caused a steric interference. Hence, in order to relieve this potential interaction the loop containing this residue was forced outwards.
These structural changes also resulted in an alteration and presumed weakening of the hydrogen bonding network in the immediate vincinity of the mutated residue. In this example, the thermal destabilization is believed to result from a combination of the all altered structural features which presumably weaken the folded form of the enzyme.
Utilization of site—directed mutagenesis in the context of protein engineering has also been applied to this project. The crystal structure of another temperature—sensitive mutant, Thrl57-»Ile, produced by random mutagenesis showed that the substitution of the
7—hydroxyl of the threonine with the increased hydrocarbon chain of the isoleucine disrupted the local hydrogen bonding network. This appeared to be the main reason why this mutant lysozyme was less stable than the wild—type enzyme (Grutter et al., 1987).
However, in order to understand how Thrl57 contributed to the stability of the T4 lysozyme, 13 different amino acids were substituted using site—directed mutagenic techniques and the crystal structures and stabilities of the resulting mutant enzymes were determined (reviewed by Matthews, 1987; Alber et al., 1986, 1987b; Alber and Matthews,
1987). The results confirmed that hydrogen bonding interactions were the main way in which Thrl57 contributed to stability. Of special note though was the crystal structure of
'pseudo-revertant' Thrl57-»Gly mutant which showed a water molecule occupying the previous position of the 7—hydroxyl group of the threonine. This water maintained the original hydrogen —bonding network giving rise to a protein whose stability was close to that of wild—type.
To date, approximately 25 different temperature—sensitive mutations have been identified at 20 different sites in the T4 phage lysozyme protein. According to Alber et al. (1987a) all of the mutations alter amino acid side chains at sites with low mobility
(with lower than average crystallographic thermal factors) and low solvent accessibility in the folded protein. Destabilizing mutations appear to interfere with specific favorable interactions, such as the van der Waals' interactions and the hydrogen bonding networks 15 discussed earlier. Alber et al. (1987a) further suggest that proteins can be stabilized by adding new interactions to regions that are rigid or buried in the folded conformation.
The feasibility of this theory is discussed in section 1.2.2C.
1.2.2B Enhancing Stability: Disulfide Bonds
Protein engineering has also been applied to the development of rational approaches which can control the resistance of a protein to inactivation due to thermal or chemical denaturation. A three-dimensional structure in these studies has been essential for modelling, in particular, the potential interactions and perturbations of proposed substitutions. However, as is discussed throughout the remainder of this section, the success of such studies seems to primarily depend upon one's ability to define the molecular basis of the denaturation/destabilization phenomenon under examination.
One of the earliest set of studies to be initiated was the examination of the role of disulfide bonds as stabilizing elements for the folded form of a given protein. As pointed out by Wetzel (1987) in his recent review, calculations based on polymer theory suggest that the fundamental basis of stabilization by a crosslink, such as a disulfide bond are in its effects upon the unfolded state of a protein structure (Schulz and Schrimer, 1979).
Specifically, a crosslink decreases the degrees of freedom available to the unfolded state thereby lowering the entropy of the unfolding process. This, in theory, should then shift the thermodynamic equilibrium between folded and unfolded states towards the former.
Studies of a genetically engineered disulfide bond in T4 lysozyme (Perry and Wetzel, 1984) and subtilisin BPN' (Pantoliano et al., 1987) not only demonstrated longer enzyme lifetimes at elevated temperatures but suggested that the enhanced stability was probably due to an unfolding restraint introduced by the mutations which reduces the proportion of unfolded enzyme. In both studies the choice of the cysteine mutations was based upon modelling studies which searched for a pair of amino acids with acceptable geometries and minimal steric constraints for the disulfide bond formation.
In some cases, the availability of a crystal structure has provided additional information regarding the origin of disulfide bond stabilizations. For example, in contrast to the studies described above, introduction of a disulfide bond into dihydrofolate reductase 16 did not confer a greater resistance to thermal denaturation (Villafranca et al., 1983;
Villafranca et al., 1987). This mutant did, however, display an enhanced stability with respect to unfolding for chemical denaturation by guanidine hydrochloride and significant changes in the folding/unfolding pathway. In addition, both the reduced and oxidized forms of this mutant were determined. Neither structure showed any significant mainchain conformational changes or atomic displacements greater than 0.25A anywhere in the molecule as compared with the wild—type structure. Comparison of the two forms of the mutant showed that the only major conformational adjustment was a simple twisting of the
C a—C/3 bond of one of the cysteine residues (Axi = 82°) in order to form a disulfide bond with a left-handed spiral geometry. The oxidized form of the mutant also displayed increased thermal mobility in a section of mainchain adjacent to the disulfide bond suggesting an ’unexpected* geometric flexibility of this bridge structure in the folded form of the protein molecule.
In a different stability study, two disulfide bridge mutants (between residues 22 and
87 and residues 24 and 87) of subtilisin from Bacillus amyloliquefacians were constructed to investigate the theory that the primary cause of irreversible thermal activation was autolysis of the enzyme (see Wells and Powers, 1986). Construction of these mutants was again based upon modelling studies of the wild—type structure which searched for ideal disulfide bond geometries with minimum steric constraints. In this case the oxidized forms of each mutant did not improve the autolytic stability when compared to the wild—type enzyme. However, comparison of oxidized and reduced forms of each mutant did show an increased stability with regard to the former. Crystal structures of the oxidized forms of these mutants showed that both disulfide bridges exhibited atypical sets of dihedral angles compared to those for other reported proteins with only minor changes in the local mainchain conformation (Katz and Kossiakoff, 1986). The only correlation between the two studies was that the mutant (24/87) which appeared to have a higher calculated dihedral energy also had the poorestautolytic stability in comparison to that of the other disulfide mutant and the wild—type enzyme. It is also interesting to note that Pantoliano et al.
(1987) did observe an enhanced thermal stability with their 22/87 mutant whereas Wells and Powers (1986) did not. The latter study speculated that a possible origin for the 17 discrepancy could be the presence of a third mutation in the Wells and Powers (1986) mutant but this claim remains unsubstantiated for the moment.
1.2.2C Enhancing Stability: Alternative Approaches
Aside from the 'general' introduction of disulfide bonds into a protein, other approaches to thermal/chemical stabilization have been attempted. All described here target upon a specific structural feature which is believed to reduce stability. For example, studies which aimed to increase the thermostability of yeast triose—phosphate isomerase
(Ahern et al., 1987) were based upon the observation that inactivation of the enzyme at elevated temperatures was believed to be related to the deamidation of asparagine residues.
The refined crystal structure of this dimeric enzyme revealed that 3 of each subunit's 12 asparagine residues were present at the interfacial van der Waals' surface. Conversion of these asparagines to charged aspartate residues would cause unfavorable interactions thereby promoting the dissociation of the dimer into catalytically inactive monomers. Replacement of two of the interfacial asparagines with smaller uncharged residues doubled the half-life of the enzyme at 100°C. Furthermore the theory regarding the origin of destabilization was tested through mutagenic replacement of one of these asparagines with an aspartate residue. The resulting mutant not only displayed decreased thermal stability relative to wild—type but an increased conformational instability and susceptibilty to proteolysis.
Matthews et al. (1987) attempted a more general structural approach to the stabilization of protein which utilized amino acid substitutions that decreased the configurational entropy of the polypeptide backbone. This involved selective replacement of a glycine residue, which has the greatest backbone flexibility, with alanine or introduction of a proline residue which has less configurational entropy than any other amino acid. In either case mutations of the form Gly-»Xaa and Xaa-»Pro must not increase entropic contributions therefore it is essential to select substitution sites which will not interfere with the three-dimensional structure of the protein. In theory, such changes should decrease the entropy of unfolding and thereby increase thermostability. In this study reversible unfolding experiments of two such mutations of T4 phage lysozyme displayed an enhanced thermal stability (higher melting temperatures) attributed to a decrease in entropy 18 rather than a change in enthalpy. In addition, crystal structures of these mutants were determined and both showed a nearly identical backbone configuration compared to the wild—type structure. This information provided necessary experimental justification for the assumption that structural changes associated with the substitution would be negligible for the approach to succeed.
A third study by Wigley et al. (1987) using the Bacillus stearothermophilus lactate dehydrogenase aimed to enhance protein thermal stability by reducing the area of hydrophobic surface in contact with the aqueous enviroment. Given the coordinates of a homologous enzyme a solvent accessible surface was generated and a hydrophobic isoleucine residue was identified as being partially exposed in the water accessible active site of the enzyme. Substitution of this residue with an asparagine resulted in a mutant which displayed a 4.5—fold increase in the half-life of the tetrameric form of this protein at an elevated temperature (90°C). However, the strength of this result is weakened by the fact that the mutant enzyme displayed altered kinetic properties including a decreased affinity for one of its substrates. In addition, the authors state that the new asparagine could potentially hydrogen bond to another uncharged polar group in the active site and this could be responsible, at least in part, for some of the observed stability. In this example a model structure was necessary for the original mutational design; however, the lack of a three-dimensional structure for this mutant prevents adequate interpretation of the origins of enhanced stability.
1.2.2D Protein Folding: Proline Isomerization
Protein engineering has also been applied to this ’more basic' level of research in an examination of proline isomerism in staphylococcal nuclease (Evans et al., 1987). This study highlights the fact that essential structural knowledge for the interpretation of designed changes is not limited to crystalline state.
*H—NMR studies of staphylococcal nuclease had previously indicated the presence of two distinctly folded conformations in solution which interconvert between each other without passing through an unfolded state. The aim of this study was to determine the structural origin of this conformational multiplicity through the examination of the 19
cis—trans isomerization of a proline peptide bond with its preceding residue (a
phenomenon known to give rise to multiple unfolded forms of proteins). The *H —NMR
studies had indicated that a particular histidine (Hisl21) was the most spectroscopically
sensitive aromatic residue to isomerism in the folded state. Furthermore, the crystal
structure of the nuclease had shown that only one of its six prolines appeared as the less
usual cis isomer. This proline (Proll7) was located only four residues away from the
'sensitive' histidine and was thus chosen as the target for the site—directed mutagenic
change to a glycine. The resulting ^H—NMR spectra of the folded Proll7->Gly nuclease
mutant did not indicate the presence of a distinguishable alternative conformation in
solution. In addition, NMR studies of the unfolded mutant protein indicated that Hisl21
displayed only one type of conformational state as opposed to the two previously observed
with the wild—type protein. These observations strongly suggested that proline isomerization
of residue 117 was a reasonable explanation for the presence of the two folded forms of
the enzyme. Interestingly, the temperature dependence of the ^H—NMR spectrum indicated
that the thermal stability of the protein had been increased by approximately 5°C but no
speculation regarding the origin of this observation was made in this paper.
1.2.3 Ligand Binding and Catalysis
The application of protein engineering to determine the functional significance of
particular residues in a binding/catalyic event dates back to the original mutagenic studies
on tyrosyl—tRNA synthetase (Winter et al., 1982) and 0 —lactamase (Dalbadie—McFarland
et al., 1982; Sigal et al., 1982). However, full exploitation of a such a project is only possible with a structure from which one can model and then later examine, if possible, designed structural changes. A three-dimensional structure was available in the case of the tyrosyl —tRNA synthetase and has been one of the essential elements for the varied studies on this enzyme over the last six years (reviewed by Fersht, 1987). With regard to ligand binding and specificity, rational protein engineering studies which seek to define and possibly alter these properties cannot do so without a model from which to base designed changes. As is discussed throughout the remainder of this sub—section, the role of the model is indispensible and when combined with additional structural characterization, in the 20 form of x-ray crystallographic or NMR studies, can provide a powerful insight into the precise chemical nature of the interactions of proteins with their specific ligands and/or substrates.
1.2.3A Ligand Binding
Protein engineering has also been applied to one of the most widely studied proteins, hemoglobin (Hb), in an effort to understand more about the specific interactions which determine the oxygen binding properties of this molecule. The particular study discussed here attempts to define, with the aid of x—ray crystallographic techniques, the functional significance of certain residues which alter oxygen affinity. Luisi et al. (1987) chose to examine the structural determinants which are responsible for the Root (Bohr) effect.
Specifically, some Hb's are particularly sensitive to allosteric modulation of oxygen affinity by protonation, including teleost fish Hb. A comparison of the amino acid sequences of the teleost fish and human Hb appeared to indicate that a single atom replacement was responsible for the functional divergence. Two mutants were constructed which compared effects related to the replacement of Cys93 — 0 with a serine residue. Both showed a marked increase in oxygen affinity consistent with the properties of the fish Hb but a decreased alkaline Bohr effect. The crystal structures of these mutants indicated that the seemingly conservative cysteine to serine substitution disrupted the normal salt—bridge formation of a neighboring residue thereby destabilizing the T —state (ligand —free) and reducing the alkaline Bohr effect. Additional mutations which could compensate for this structural perturbation were also constructed and in the most favorable case not only maintained an enhanced oxygen affinity but restored the alkaline Bohr effect to a level
60% larger than human Hb. The crystal structures of these new mutants are in preparation.
Sligar et al. (1987) have used protein engineering studies to examine the coordination geometry of a prosthetic group in cytochrome 65. In this study the three-dimensional structure of core domain of this protein was known from X—ray crystallographic and NMR investigations. The active center of cytochrome b$ is a heme group ligated to two histidine side chains. From the known geometries of these heme axial ligands it was postulated that 21
substitution of one of the histidines with a methionine might generate a protein whose
coordination geometry mimicked that of high spin six—coordinate cytochromes c and ^>552*
Resonance Raman spectra demonstrated an altered spin state for the His-»Met mutant
similar to that observed for the c and £552 proteins. Substitution of the histidine was also
expected to open the heme binding cleft for additional ligand coordination. This hypothesis
was believed to explain the enhanced peroxidase activity observed for this mutant
compared to the wild—type protein. The most interesting aspect of this study was that this
mutant acquired a novel activity for hydrogen peroxide dependent oxidative demethylation.
Because of the low spin state of the wild—type molecule such activities are not observed.
A more detailed interpretation of all the properties listed here must await further studies.
NMR methods have also been utilized as part of protein engineering studies which, in
this example, examine the binding of the Ca^+ ion in bovine calbindin Dg^ (Linse et al.,
1987). The crystal structure of the molecule has shown that the two calcium binding sites
(I and II) are composed of two helices separated by a loop containing approximately 12
residues. Mutations were made in site I to probe the effects of localized structural changes
in the loop upon Ca^+ ion binding by removing a proline (mutant M3) or replacing it
with a glycine and removing another liganding group (mutant M2). A third type of
mutation (M4) was made which attempted to perturb the stability of the helix—loop—helix
structure. The ^H—NMR spectra of all mutants in the study showed no major changes in
the overall tertiary structures compared to the wild—type protein. Mutants M2 and M3
both demonstrated a considerable reduction in Ca? + affinity for site I (from an exchange
rate of 3s— * to 5000s ” 1) with no effect upon the affinity of site n. From these results it was then possible to make assignments to specific aromatic residues which were involved in ligand binding in site I. Mutant M4 which aimed to probe the stability of the binding structure demonstrated no differences in its affinity for Ca^+ compared to the wild—type protein but did display altered thermal stability. The most structurally interesting result was the establishment of cooperative Ca^+ binding in the wild—type and M4 mutant. Because assignments had been made to one site, as previously described, the ratios between the binding constants of both sites could be estimated. A free energy of site—site interaction could then be calculated: wild—type = —5.1 ± 0.4kJmol—* and M4 < —3.9kJ mol- *. 22
Hence, the proposed structural perturbations of the M4 mutant appeared to be represented
through an unexpected decreased cooperativity between the two binding sites.
Ligand binding sites have also been engineered by introducing structural motifs or
binding domains from one protein into another. Intwo studies of the 434 repressor the
binding specificity and location of the recognition site were determined through the
exchange of structural features from two other repressor proteins: the 434 cro protein
(Wharton et al., 1984) and the P22 repressor (Wharton and Ptashne, 1985). Based upon
the structure of the 434 repressor and several other DNA binding proteins, DNA sequence
recognition is thought to be located, at least in part, in a particular a —helix. In the case
of the cro protein this helix was identified from crystallographic studies. Wharton et al.
(1984) chose to exchange this helix with what was assumed to be an equivalent structure
in the 434 repressor (no crystal structure was available at this time) to see whether this
particular feature was responsible for binding and specificity. The resulting hybrid protein
bound the cro recognition sites with the same affinity as the original cro protein.
Construction of the P22 repressor recognition site was achieved through specific amino acid
substitutions on the exposed region of the selected a —helix (Wharton and Ptashne, 1985).
As with the hybrid cro protein, the mutant 434:P22 protein exhibited the same affinity as
the P22 repressor for P22 sites. Jones et al. (1986) and more recently Riechmann et al.
(1988) also chose to exchange binding domains in a set of studies which involved the grafting of a mouse antigen binding site on to the base of a human antibody. Their aim was to produce a antibody with a specific hapten affinity which was less immunogenic to humans then one derived exclusively from a mouse source. In the earlier study (Jones et al., 1986) the crystal structure of the human antibody, human myeloma protein NEWM, had been previously determined allowing identification of the particular residues which comprised the hapten binding site. These residues were exchanged through subsequent genetic engineering experiments for the mouse antibody binding site. This hybrid mutant protein displayed a similar hapten affinity compared to the original mouse antibody protein and loss of a previously immunogenic site. Riechmann et al. (1988) have extended this work by replacing this engineered binding site into the original rat antibody to accurately identify the structural unit responsible for antigen binding. However, the engineered 23
antibody was not effective and the design fault was identified through a modelled
comparison of the antigen—antibody structure based upon the coordinates from a related
crystal structure. Subsequent alteration of two residues reestablished antigen binding.
Additional genetic engineering experiments were used to select the best human constant
chain for cell lysis and a final genetic combination of this chain with the modified binding \ site produced a 'humanized' antibody suitable for therapeutic application.
1.2.3B Identification of Active Sites: Domain Structures
The three-dimensional crystal structure of an enzyme does not necessarily insure that
one can identify the precise location of the active site. However, such structural
information in combination with site—directed mutagenesis has led to the location of
specific residues or more generally domains responsible for catalytic activity. A recent
example of this is the location of one of the active sites in the large fragment (Klenow
fragment) of DNA Polymerase I (Joyce and Steitz, 1987). Specifically, the crystal structure
of the Klenow fragment showed dNMP (deoxynucleoside monophosphate) bound to the
smaller of the two domains. dNMP is a known inhibitor of the enzyme's 3'->5' exonuclease
activity and its position in the active site was assumed to mark the position of the
phosphodiester bond that would be cleaved in the exonuclease reaction. In addition, two
metal binding sites (A and B) were located near the dNMP molecule with one of the
metal ions (A) coordinated to the 5' phosphate of the dNMP. Two mutants were then
constructed involving amino acid substitutions at each metal binding site. In both mutants
exonuclease activity was abolished but polymerase activity (the second activity of this
enzyme fragment) remained unchanged. Crystallographic analysis of both mutants were
performed and indicated that neither had undergone any large conformational changes.
Mutation of metal site B yielded protein crystals which bound dNMP and a divalent cation
in metal site A but no cation in metal site B, suggesting that this metal ion (B) played a
direct role in catalysis. Mutation in metal site A prevented binding of both cations and
dNMP in the crystals thereby suggesting that this site was involved in substrate binding
and possibly catalysis. This information taken together with the small structural pertubations observed suggested that the 3'->5' activity was indeed located in the smaller domain and 24 implied that the polymerase active site was probably separated from this site to some degree.
Protein engineering studies on ribulose—biphosphate carboxylase/oxygenase (rubisco) from Rhodospirilium rubrum have utilized site—directed mutagenesis to determine whether each of the two subunits contained an independent active site or whether active sites were created by intersubunit interactions (Larimer et al., 1987). Crystallographic and chemical crosslinking studies had indicated an interaction between the N—terminal region of one subunit and an essential lysine residue of another. Two mutation sites were then selected, one altering the essential lysine and one altering a glutamate residue located approximately
9A from the lysine residue. Both mutations abolished activity but still allowed substrate to bind. Expression of LysrLys or Glu:Glu homodimer mutants showed no activity as expected. Expression of LysrGlu heterodimers, however, displayed half of the wild —type activity (normally two active sites per dimer) indicating the successful formation of one active site thereby demonstrating the presence of a shared active site between subunits.
In a third example, the crystallographic studies of tyrosyl—tRNA synthetase (TyrTS; see Chapter 4) from Bacillus stearothermophilus failed to show the presence of a 'domain' comprised of the C —terminal 99 amino acids in the enzyme (419 residues total).
Construction of a mutant which lacked this C—terminal domain (truncated form) produced a mutant which could no longer bind the tRNA substrate necessary for the second catalytic step (charging of tRNA) of its two-step reaction mechanism (Waye et al., 1983).
However, this dimeric truncated mutant still demonstrated 'half—of—the—sites activity'
(Fersht et al., 1975a). Specifically, in the first step of this reaction one mole of tyrosyl adenylate is formed in only one of the wild—type and the truncated enzyme's two active sites. Construction of heterodimers (one full-length: one truncated enzyme) which contained mutations that prevented catalysis of the first step of the reaction demonstrated that the tRNA molecule bound to the subunit which did not form tyrosyl adenylate in its active site. This study also suggested that the acceptor arm of the tRNA had to extend across to the opposite active site in order to become charged in the second stage of the reaction (Carter et al., 1986). Bedouelle and Winter (1986) used this information in combination with site—directed mutagenesis across the known surface of the molecule to 25 produce a model of the binding of the tRNA to the enzyme. In this example several protein engineering studies provided a means for deriving a liganded model from an unliganded structure.
1.2.3C Probing the Energetics of Enzyme Catalysis
Understanding a particular enzyme reaction as stated by Fersht (1987) relies on the one's ability 'to characterize the bound substrates, intermediates, products, and transition states on the reaction pathway [and] to determine the interaction energies between them and the enzyme as the reaction proceeds.' In his review of the protein engineering studies on the tyrosyl —tRNA synthetase (TyrTS) from Bacillus stearothermophilus Fersht (1987) describes how the mechanism of activation of the tyrosine substrate with ATP, the first step of the two-step reaction (see previous section 1.2.3B), has been elucidated. The end product of this reaction is an enzyme —bound tyrosyl adenylate complex. The stability of this product on the enzyme has allowed a crystal structure of this complex to be obtained
(see Chapter 4). Detailed kinetic analyses have also allowed the determination of individual rate and binding constants along this particular reaction pathway (see Chapter 3). The initial studies on this enzyme aimed to determine the energetic contributions of particular side chains, identified from the crystal structure to interact with the tyrosyl adenylate, to binding and catalysis. This was acheived through systematic substitutions of potential hydrogen bonding donor ar acceptor groups on the enzyme using site—directed mutagenesis techniques. Subsequent comparison of the kinetics of tyrosine activation between the wild—type and each mutant protein provided a means of measuring of the relevant free energy values (Winter et al., 1982; Wilkinson et al., 1983). Detailed examples of different kinetic experiments on this enzyme may be found throughout the remainder of this thesis and will not be discussed in any greater detail here (see Chapters 3 and 5, in particular).
The articles reviewed below describe studies of two other enzymes in which similar types of experiments have been attempted.
A successful determination of the reaction energetics of a mutant triosephosphate isomerase (TIM) have also been achieved (Raines et al., 1936). As with the TyrTS enzyme described above the three-dimensional structure of TIM with its natural substrate 26
had been determined. The rates of the individual steps in the reaction catalyzed by this
enzyme had also been determined. Mechanistic work had demonstrated that the abstraction
of a proton in the first step of the reaction to yield a cis—enediol intermediate was
probably effected through the carboxylate group of a glutamate residue (Glut 65) located
within the active site of TIM. Using site—directed mutagenesis methods this residue was
replaced with an aspartate. The catalytic activity of the resulting mutant proved to be
1000—fold less than that of the wild—type enzyme. Furthermore, examination of the
individual steps of the reaction showed that the mutant specifically destabilized the
transition states of the two intermediate enolization steps in this four step reaction. Hence,
this study has been able to verify the essential role of a residue predicted to participate in
catalysis from a structure of the enzyme complex. However, the structural alterations,
which might have resulted as a consequence of this mutation, could not be considered. A
full interpretation of kinetic results requires a structural analysis of this mutant.
The example of the recent study of mutants of staphylococcal nuclease (Hibler et al.,
1987) further emphasizes the point that precise structural information, in solution as well
as the crystalline state is necessary for a proper interpretation of the effects of
site—directed alterations upon catalysis. The crystal structure of the nuclease had suggested
that the carboxylate group of a glutamate residue (Glu43) located in the active site was
directly involved as a general base in its catalytic reaction. Five different amino acid
substitutions, including an aspartate, were made at this position using site—directed
mutagenic techniques. The steady—state parameters for the enzyme hydrolysis indicated that
the activity was reduced from 1400—fold in the aspartate mutation to 5000—fold for the other charge—neutral mutations. Comparison of these values with the nonenzymatic reaction rate for hydrolysis of nucleotides suggested that some catalytic effects persisted when the carboxylate was removed. In addition, thermal denaturation studies of all five mutants showed that all displayed an enhanced thermal stability compared to the wild—type molecule. This suggested the possibility that significant structural changes may have occurred to the folded form of these mutant enzymes which was verified in subsequent one— and two-dimensional *H—NMR experiments. The spectra for two mutants Glu43-»Asp and Ser were presented and compared with the wild—type spectra. In 27
general, the ligand—free forms of both mutants showed 'modest' spectral changes. In the
presence of activesite ligands the changes were more pronounced suggesting
conformational changes from 0.4 to 0.6X located at least 15X from the site of the
mutation. Computer modelling studies suggested that the origin of the changes (chemical shifts) was related to alterations in solvent accessibilities of the residues located near the
mutation. It was postulated that the substitution of smaller residues at position 43 could alter the position or magnitude of buried charge with consequences which could in turn propagate throughout the structure. However, the principle conclusion of this paper was that the relationship between the observed conformational changes to the kinetic properties of the mutants is unknown and further interpretation demands additional high resolution structural analysis.
1.2.3D Specificity
The area of protein engineering where the need for a model structure is most obvious is the design of alterations to an enzyme to probe questions of specificity. An early example of this involved the attempt to redesign substrate specificity in rat pancreatic trypsin II (Craik et al., 1985). Using the known three-dimensional structure of a related enzyme, bovine cationic trypsin, computer modelling studies of the active site suggested that mutation of two glycines to alanine could differentially affect the binding of arginine and lysine substrates to the enzyme. In the first case, Gly216-»Ala, a water molecule necessary for the binding of a lysine side chain but not an arginine side chain should be displaced resulting in an enhanced specificity towards arginyl substrates. The results showed a shift in the specificity constants towards arginine but the origin of the differences was related to a reduction of kcat as well as changes to suggesting alterations to the mode of binding and the positioning of the scissile bond of lysine substrates. The second mutation, Gly226—>Ala, was expected to display enhanced specificity towards lysine substrates because the new methyl group in this instance was supposed to sterically hinder the binding of the longer arginine side chain. The resulting 20—fold shift in specificity for lysine was also accompanied with a reduction in kcat. The double mutant (Gly216,
Gly226—>Ala) also displayed an affinity preference for lysyl substrates but in this case the 28 mutant enzyme exhibited almost no catalytic activity. Analysis of all mutant enzymes by native gel electrophoresis indicated changes between the wild—type and the 226 and double mutant which suggested that a major conformational change in these enzyme structure could have occurred. As has been pointed out in previous sections of this chapter, modelling studies of a known structure provided the means for the designed alteration but interpretation of the resulting effects will also rely on as detailed high resolution analysis.
In this example the modification of specificity could not be effected without poorer substrate affinities and reduced catalytic activity. This work provides a clear example of the inseparable nature of binding and catalysis.
A novel approach to engineering enzyme specificity was described by Carter and
Wells (1987) which was based around the removal of a catalytically essential group from the active site of subtilisin. Modelling studies had shown that a histidine in a bound substrate could be superimposed on the enzyme’s active site histidine which was essential for the catalyic cleavage of a peptide bond. The working hypothesis assumed that removal of this group (by a His-»Ala mutation) should result in an enzyme which can only cleave peptide bonds when a histidine is present at the appropriate position in the substrate to restore the enzyme's catalytic activity. Such a phenomenon was termed 'substrate—assisted catalysis.' Experiments using substrates which contained the normal wild—type specificity determinants demonstrated that the presence of histidine at the required position in the peptide fragment resulted in a 400 times faster hydrolysis rate compared to substrates with
Ala or Gin in the corresponding position. Compared to the wild —type enzyme, hydrolytic activity was partially restored using the histidine containing substrate.
In this third example substrate—specificity properties have been specifically recruited from one type of subtilisin into another (Wells et al., 1987). Specifically, two variants of subtitlisin demonstrate a broad specificity towards hydrolysis of peptide chains. However, with glutamate at the PI position subtilisin from Bacillus lichenformis is 10 times as catalytically efficient at degrading particular peptide sequences compared to subtilisin from
Bacillus amyloliquefaciens. Sequence comparisons and modelling studies using the structure of the latter enzyme suggested that the replacement of three of its residues could enhance its specificity to substrates with glutamamte at the PI position. The resulting triple mutant 29
of B. amyloliquefaciens verified this 'structurally—based' prediction by demonstrating
activities that approached those of subtilisin from B. licheniformis.
1.2.4 The Conformation of Polypeptides: Structural Elucidation
The aim of this literature review was to show the necessity of a three-dimensional
structure or model for the design and interpretation of protein engineering experiments.
However, the elucidation of structure, a completely separate field of study, has also been
able to take advantage of the methods of site—directed mutagenesis. For example, the ab
initio determination of a three-dimensional crystal structure of a protein often relies upon
the production of isomorphous heavy—atom derivatives which can provide the necessary
phase information needed for solving the structure. Crystallized proteins, however, may not
react with heavy—metal compounds in one instance or alternatively may react but create
derivatives that are too complex for interpretation or are even non —isomorphous with the
parent compound. Dao—Pin et al. (1987) have attempted to explore the feasibility of
generating heavy—atom reaction sites on a protein using a set of five different
cysteine—containing mutants of phage T4 lysozyme. The two cysteine residues in the
wild—type version of this protein were known to react with mercuric chloride to yield
excellent isomorphous derivatives. Each of the mutants constructed contained a third
cysteine residue located on the surface of the molecule. All mutants were crystallized,
soaked under the appropriate conditions to effect the reaction of the mercuric ion and the
structures were determined. In general, cysteines which appeared to be the most exposed
and most mobile appeared to be the least reactive. The best mercuric derivatives were
obtained from partially exposed cysteine side chains, located for example in surface
crevices. This study then provides general guidelines for engineering such heavy—atom sites
and also serves the first systematic study of its kind.
The refinement of the crystal structure of tyrosyl—tRNA synthetase has also been
aided using the truncated form of the molecule described previously (section 1.2.3B). As
was noted earlier, the final 99 residues of enzyme appeared disordered in the full-length
wild—type crystal structure. Refinement of this structure relied on a model which used calculated structure factors for this uninterpretated electron density and was reflected in a 30
large crystallographic R—factor, 32%. The structure of the truncated mutant, however,
could be determined to a resolution of 2.5X reolution with an R —factor of 18.7% using
molecular replacement methods based on the original wild—type structure (Brick and Blow,
1987). The more accurate phases obtained for this known portion of the protein were then
used to further refine the original wild—type structure thereby reducing its crystallographic
R —factor to 23% (P. Brick, personal communication; see also section 4.1.3).
Site—directed mutagenesis studies have also been utilized for the sequence—specfic
assignment of resonances arising from *H —NMR spectra. As was discussed in section
1.2.3A, the assignment of resonances to one of two ligand binding sites in calbindin
arose as a consequence from engineered differential affinities for the Ca^+ ion (Linse et
al., 1987). In an earlier study, Gronenborn et al. (1986) was able to make
sequence—specific assignments to aromatic group resonances of human interleukin—1/3 using
specially constructed site—directed mutants. In that study, tyrosine residues were mutated
to phenylalanine and the only histidine residue in the molecule was changed to an
asparagine, ^identification of each aromatic resonance was done by comparing mutant and
wild—type ^H—NMR spectra. The absence of a resonance in the mutant spectrum was used to indicate the assignment of a sequence—specific resonance at the corresponding position in the wild—type spectrum. A similar strategy has also been used for the assignment of histidine resonances in the ^H—NMR spectrum of subtilisin (Bycroft and
Fersht, in preparation).
1.3 Project Aims
As is evident throughout the previous section, protein engineering studies have consistently shown that one can alter the functional properties of a protein by selectively mutating a single amino acid. The primary aim of the research presented in this thesis is to understand the relationship between observed changes in catalytic function and corresponding changes in the three-dimensional structure in the enzyme tyrosyl—tRNA synthetase (TyrTS) from Bacillus stearothermophilus. The emphasis of this work is on the structural interpretation of the catalytic effects of selected mutants of the TyrTS using
X—ray crystallographic methods. 31 Residue threonine 51 of the TyrTS had been selectively changed to a proline (mutant
TP51) using site—directed mutagenic techniques. The resulting enzyme demonstrated an enhanced catalytic activity for the formation of tyrosyl adenylate (the product of the first step of the reaction of the TyrTS): a 25—fold increase compared to the wild—type version of the enzyme (Wilkinson et al., 1984). In order to understand how the enzyme had been
'improved* it was necessary to see if and how its structure had been altered by the introduction of the proline side chain. This was done by determining the three-dimensional structure of the TP51 mutant. In addition, the crystal structures of three other mutants at position 51 (Ala, Gly and Ser) are also presented in order to correlate general trends of observed catalytic activity with common or systematic changes in structure. However, all structural results (including the crystal structure of the TP51 mutant) emphasize the importance of the chemical nature of the altered side chains as opposed to gross structural rearrangements of the mainchain.
With respect to the kinetic data, these crystallographic results suggest that the reason for enhanced activity of the TP51 mutant is related to a significant contribution of apolar interactions between its side chain and tyrosyl adenylate. This expanded the original hypothesis which suggested that the origin of the enhanced activity of the TA51
(Thr51-»Ala) and TP51 mutants was due, at least in part, to the loss of a poor hydrogen bond between the tyrosyl adenylate and the hydroxyl group of the wild—type threonine residue (Wilkinson et al., 1984). The significance of the presence of hydrophobic groups could be tested by substituting valine at position 51 which contained an additional methyl group compared to the wild—type threonine side chain. The genetic construction and the subsequent kinetic characterization of this mutant are presented to examine the validity of this claim.
Localized entropic changes which may potentially arise from the introduction of a proline side chain into the TyrTS structure may also contribute to the enhanced activity displayed by the TP51 mutant. The theoretical considerations of this hypothesis with regard to its testability through the construction of another proline containing mutant are discussed. Included as well are the specific details of design of such a mutation which focusses upon the 'rules' regarding the placement of proline residues in helical structures. 32
Finally, the predicted structural alterations of mutant His48—>G ly (HG48) are examined. In this particular case, kinetic studies suggested that a structural interaction existed between the histidine residue at position 48 when proline was present at position
51. Evidence of such an interaction is investigated through the crystal structure determinations of both the TP51 and the HG48 mutants. The structure of the HG48 mutant is also used to examine the feasibility of a proposed mechanism for its enhanced hydrolytic degradation of the enzyme—bound tyrosyl adenylate complex. 33
CHAPTER 2
OLIGODEOXYNUCLEOTIDE-DIRECTED MUTAGENESIS
2.1 The Automated Synthesis of Oligonucleotides
2.1.1 Introduction
The aim of DNA synthesis is to make a sequence specific polynucleotide chain through the formation of a covalent phosphodiester linkage between the 3' carbon and the 5' carbon of adjacent deoxyribose nucleosides. The two most widely used synthetic techniques are the phosphotriester method (Sproat and Gait, 1984) and the phosphite triester method (Tanaka and Letsinger, 1982; Atkinson and Smith, 1984). Because monomeric nucleosides have two free hydroxyl groups (3' and 5') both methods demand the chemical protection of one group to ensure that sequential nucleoside addition occurs with the 3’->5’ or 5'-*3' linkage (not 3'-»3\ for example). This is generally achieved by protecting the 5' hydroxyl of each nucleoside and phosphorylating or phosphitylating the 3' hydroxyl group.
The first step of automated synthesis involves the removal of the temporary protecting group on the 5( hydroxyl of the nucleoside bound to the solid support. This step is often called "detritylation" because the dimethyltrityl group is commonly used for such protection. The phosphorylated or phosphitylated 3* hydroxyl group of the next nucleoside in the required sequence is activated and added to the free 5* hydroxyl of the bound nucleoside. This is the coupling step. In the capping step, any unreacted
5' hydroxyl groups are irreversibly modified (by acetylation) to prevent further nucleoside addition in subsequent steps. Additionally, if the phosphite triester method is used then an oxidation step is required which changes the unstable trivalent phosphite triester (succeptible to acid and base cleavage) to a stable pentavalent phosphotriester.
The cycle continues until the nucleotide sequence is completed. Finally, the oligonucleotide is cleaved from the support and fully deprotected.
The automation of DNA synthesis has been primarily influenced by the development of solid support synthetic techniques and improved coupling chemistry.
The Applied Biosystems DNA synthesizer (Model 380B) uses a modified version of the
phosphite triester method of oligonucleotide synthesis called the phosphoramidite method
(Beaucage and Caruthers, 1981). The phosphoramidites are chemically modified nucleosides which serve as the monomeric units of addition during the synthesis.
Specifically, the dimethylamino group replacing the traditional chloride in the nucleoside monomers has improved stability (resistant to aqueous hydrolysis and air oxidation) and reactivity (controlled by the addition of weak acid). It follows that coupling efficiencies using phosphoramidites are very high (98—100%) which allow for the successful synthesis of long oligomers (up to lOOmers) and occassionally the use of short oligomers (15—25mers) without additional purification (Sanchez—Pescador and Urdea,
1984).
2.1.2 Materials
(a) Monomers
Nucleoside monomers were obtained as dried powders (0.5 gram) stored under argon
from Applied Biosystems and were dissolved in anhydrous acetonitrile shortly before synthesis commenced:
N—6—benzoyl—adenine (A^) phosphoramadite
N—4—benzoyl—cytosine (C^2) phosphoramadite
N—2 -iwbutryl—guanine (G*b) phosphoramadite thymine (T) phosphoramadite
(b) Support
A^z, C^z» and T controlled pore glass (CPG) columns (pore diameter 500A, particle size 125 to 177 microns in diameter) were used depending upon the nucleotide at the 3' end of the mutagenic primer. Each deoxyribonucleoside (0.2 micromoles) was esterified to the end of a long organic linker which was attached to the CPG via a siloxane bond. All silanol groups on the linker and amino groups on the nucleoside were appropriately capped or protected. 35
(c) Chemicals and Solvents
All chemicals and solvents for automated synthesis were supplied by Applied
Biosystems.
Analytical grade ammonium hydroxide for final deprotection (sp. gr. 0.880, 35% in water) was supplied by BDH Chemicals, Ltd..
2.1.3 Method
(a) Syntheses
All mutagenic oligonucleotides were synthesized by Mr. Jack Knill—Jones using an
Applied Biosystems automated DNA Synthesizer Model 380B following a standard
(Applied Biosystems software) cycle.
(b) Final Deprotection
1 —2ml of ammonia were added to the DNA collection vial containing the oligonucleotide which was then sealed and wrapped in Parafilm. The vial was placed in a 55°C water bath overnight (minimum 6hr). The sample was then dialyzed against deionized water overnight at 4°C and stored at — 20°C.
(c) Spectrophotometric Analysis
The concentration of the oligonucleotide was calculated from its absorbance at 260nm using Beer's Law:
A = Em x c x 1
A = absorbance at 260nm; Em = extinction coefficient of the oligonucleotide; c = concentration; 1 = path length
Em was estimated using the following nucleotide extinction coefficients, em's: 8.8, 7.3,
11.7, and 15.4/iM- ^cm“ * for T, C, G, and A respectively. No allowance was made for hypochromicity effects, hence the calculated concentration may have been a slight underestimation of the true concentration. The peak absorbance and a spectrophotometric scan (200nm to 300nm) of each oligonucleotide was measured using a Perkin—Elmer Lamba 5 UV/Vis spectrophotometer. 36
2.2 The FPLC Purification of Oligonucleotides
2.2.1 Introduction
Although automated synthesis of oligonucleotides often yields a product capable of experimental use without further purification, single step purifications are routinely carried out to ensure a reliable standard of purity. All purification techniques aim to isolate the desired product from impurities which occur during synthesis. These include truncated chains from incomplete coupling and base modified chains or cleaved chains arising from exposure to deprotection reagents.
The Pharmacia FPLC (Fast Protein Liquid Chromatography) system was exclusively utilized for purifications of all mutagenic oligonucleotides presented as part of this thesis. The aim of the system is to provide a simple high speed and high resolution method of column separation. For strong anion exchange, the support is typically derivatized with quaternary amines. The oligonucleotide is usually fully deprotected to allow separation based on net charge differences arising from charged phosphate and base groups. The sample mixture containing the oligonucleotide is fully bound to the column then selectively eluted using a buffered gradient of increasing ionic strength.
2.2.2 Materials
(a) Support
A Pharmacia Mono Q HR 5/5 column (5mm x 5cm glass column) packed with 1ml
Mono Q MonoBeads (particle size 10/im; loading capacity for peak, 5mg) was used.
The Mono Q column is a hydrophilic polyether support functionalized with trimethylammonium—methyl groups. It is a strong anion exchanger and is stable over a pH range from 2 to 12.
(b) Solvents lOmM NaOH and lOmM NaOH/1.5M NaCl were first filtered through a 0.45/an
Millipore (Type HA) filter to remove any particulate material before application to the
FPLC system. 37
(c) Apparatus
The Pharmacia FPLC system was used for purification of all the mutagenic oligonucleotides. The system consisted of a gradient programmer (GP—250), two dual syringe pumps (P—500), a single path UV monitor (UV—1) which monitors the absorbance at 254nm and a programmable fraction collector (FRAC—100). A column trace was produced by a two channel chart recorder (REC—482) using input from the
UV monitor.
2.2.3 Method
The Mono Q column was first equilibrated with lOmM NaOH. The oligonucleotide sample was spun for lOmin in a microfuge at room temperature to remove particulate material before application to the FPLC system. Approximately 40/xg (maximum 60/xg) was loaded on the Mono Q column. The oligonucleotide was then eluted using a linear salt gradient from 0.3 to 1.1M NaCl in lOmM NaOH. Using a flow rate of l.Oml/min, the eluate was collected in 0.5ml fractions and purification was completed in 40min.
Oligonucleotides (17—23mers) typically eluted between 0.5M and 0.7M NaCl. A representative elution profile is shown in Figure 2.1. Fractions corresponding to the major peak on the elution profile (verified by measuring their absorbance at 260nm) were pooled and desalted by dialysis against deionized water at 4°C and then stored at
—20°C until further use.
2.3 Site—Directed Mutagenesis
2.3.1 Introduction
Site—directed mutagenesis encompasses a variety of techniques which aim to make precise changes to a sequence of nucleotide bases. Since the pioneering work of
Hutchinson and coworkers (Hutchinson et al., 1978; Gillam and Smith, 1979) whereby synthetic oligonucleotides were used to effect the desired base changes, numerous strategies have since been developed (reviewed by Smith, 1985; Carter, 1986).
Before oligonucleotide mutagenesis can commence it is necessary to obtain a 38
Figure 2.1
Course of the elution (l.Oml/min)
A Typical elution profile for a 19mer oligonucleotide (YTSAP50, Table 2.1) from the
Pharmacia FPLC Mono Q HR 5/5 column. The solid line is the absorbance at 254nm.
The dashed line is the sodium chloride gradient. Fractions from the peak were collected and dialysed against deionized water. 39
single—stranded DNA fragment containing the gene of interest. The single—stranded
genome of M13, a rod—shaped bacteriophage, has been a popular choice. It is easily
propagated and isolated for mutagenesis experiments and a range of cloning vectors
exist which have been derived from its genome (Carter et al.( 1985b). However,
expression of the mutated gene often requires an excision (using restriction enzymes)
and a ligation into a plasmid vector that contains a stronger, often inducible promoter.
Alternatively, vectors have been developed using modified plasmids which are capable
of being isolated in single—stranded form. These include the pEMBL series (Dente et
al., 1983) and the pBR322—derived 'phagemids' (Studier and Moffat, 1986; Mead et
al., 1986; Zoller et al., 1987). The design of these vectors permits mutagenesis and
expression to be carried out in the same system.
Oligonucleotide—directed mutagenesis in its most simple form begins with the
annealing of the mutagenic oligonucleotide, a synthetic oligonucleotide containing the
desired base changes, to the single—stranded template. The oligonucleotide is then
extended from. its 3 ' end using the Klenow fragment of DNA polymerase I in the
presence of T4 DNA ligase to close all gaps. The heteroduplex is then transformed in
cells where it will propagate. Because all vectors go through a single—stranded phase
as part of replication the original duplex will segregate into two (wild—type and
mutant) populations. From isolated populations, in the forms of colonies or plaques,
single—stranded DNA is attached to a support filter of nitrocellulose or nylon. A
radio—labelled form of the oligonucleotide can be used to probe the DNA attached to
the filter because it preferentially hybridizes to DNA containing the the mutation
(Wallace et. al., 1980). Oligonucleotides up to 20 nucleotides long can be dissociated
from wild—type DNA by washing the filter at temperature slightly below the melting
temperature (Tjj) for perfect strand separation calculated using the 'Wallace Rule'
(Suggs et. al, 1981):
Td(°Q = 2 x number of A,T base pairs Eq. 2.2
4 x number of G,C base pairs.
The probe should remain hybridized only to those colonies containing the mutation (a perfect match) which will appear "lit" on an autoradiograph of such a filter. 40
As part of this study, three mutagenic strategies were attempted using an Ml 3 template which contained the tyrS (tyrosyl—tRNA synthetase) gene: single priming
(previously described), coupled priming with amber selection (Carter et al 1985ab), and the Kunkel method of mutagenesis (Kunkel, 1985; Kunkel et al., 1987). In the coupled priming approach with amber selection a second primer is annealed, extended and ligated concurrently with the mutagenic primer. This selection primer reverts an amber mutation located in a gene which codes for an essential phage function. The double—stranded heteroduplex is then transformed into a nonsuppressor strain of
Escherichia coli. Only phage which no longer carry the amber mutation will be able to propagate, a reasonable percentage of which should carry the intended mutagenic change. The Kunkel method on the other hand relies upon producing a template for mutagenesis which will be destroyed upon transformation. Single—stranded mutagenic template is first produced by growing in an E. coli strain deficient in dUTPase (dut) and uracil N—glycosylase (ung) which are required for the correction of uracil improperly incorporated into template DNA. Single priming mutagenesis is then carried out using this uracil—containing template. The newly synthesized strand contains no uracil. The heteroduplex is then transformed into a nondeficient E. coli strain. Ideally, original wild—type template will be destroyed leaving only template carrying the mutagenic change to be propagated; however, mutagenic frequecies which are actually achieved using this method range from 50 to 70% (Kunkel et al., 1987).
2.3.2 Materials
2.3.2A Mutagenesis Using the Single Priming and the Amber Selection Approaches
(a) Phage
Mutagenesis was performed in the tyrS gene cloned in M13mp93 (Messing and Vieira,
1982; Winter et al., 1982). In the case of amber selection the construct,
M13mp93AmIVTyrTS, carried the amber mutation (originally glutamine) in gene 4 of the Ml3 phage genome. The sequence of this selection primer and its relation to the wild—type genome were as follows (Carter, 1985): 41
3' CACTACCTATCTCAGAA 5 ' Amber IV P rim er
*
5 ’ gt & ATGCACAGACTCTT 3' W ild-Type Tem plate
mismatch (*) at position 5327 within M13mp7
The inserted segment consisted of the tyrS gene containing 1,257 bases flanked at its
5' end by approximately 1,000 bases of Bacillus stearothermophilus DNA and at its 3’ end by 296 bases, 184 of which are from the plasmid pBR322 (Winter et al., 1982).
(b) Oligonucleotides
Table 2.1 lists the sequences of the oligonucleotides and their calculated melting temperatures. The YTSTV51 (single priming) and the YTSAP50 (amber selection) oligonucleotides were synthesized and purified according to the procedures previously described in sections 2.1 and 2.2.
(c) Strains
Phage were grown for template preparation on the E. cdi K12 derivative strain JM101
(Messing, 1979). This strain possesses an episomal F factor that complements a deletion in the pro region (proline biosynthesis) of the bacterial chromosome. The F factor is not transmissible and only E. cdi cells expressing an F pilus are able to be infected by Ml 3 phage. JM101 is also supE, necessary for growth of the template containing the amber mutation. Transformation was accomplished using the repair deficient strain BMH 71—18 mutL strain (supE) (Kramer et al., 1984) with the single priming approach and HB2154 (supE~) (Carter et al., 1985b) with amber selection.
Plating cells for the transformation were JM101 or HB2151 (Carter et al., 1985b) which paralleled the phenotype of the CaClj treated cells, respectively but were not repair deficient. Stocks of strains were maintained on minimal plates supplemented with glucose (Appendix I) for selection of proline prototrophy (F factor). Colonies were restreaked every 4 weeks, grown at 37°C and stored subsequently at 4°C. Table 2.2 lists the genotypes of all strains utilized throughout the work described in this thesis. 42
Table 2.1 Primers used for mutagensis of the tyrS gene
P rim er Sequence C a lc u la te d Codon 5 '—>3' Td/°C Change
YTS17S18 GTCTT CAT CACT CGTCGTTTGGT 68 ACGGAT-> Thr17Asp18—>Thr17SerAsp18 ACGAGTGAT
YTSAP50 AAAATGGTCGGCAAGTGGC 58 GCC->CCG Ala50—>Pro50
YTSTV51 GT CAAAAT GACGGCCAAGT 56 ACC->CTC Thr51—>Val51
Table 2.2 Summary of bacterial strains
S tr a in Genotype R eferen ces
JM101 K12, A( lac-pro), supE, th i/ F* , traD 36, proA+H**, lac 19, lacZ AMI3 Messing, 1979
BMH 71-18 K12, A ( lac-pro), supE, thi/ mutL F' proA+B+, lac 1*1, lacZ AM13, m utL::TnlO Gronenborn, 1976
HB2151 K12, ara, A(lac-pro), thi/ F' proA+B+, lac IQ, lacZ C a rte r e t al., AMI 3 1985b
HB2154 same a s HB2151 but C a rte r e t al., mutL::TnlO 1985b
CJ236 dut-1, ung-1, thf-1, re/A-1, Kunkel et al., pCJ105(Cmr ) 1987 43
(d) Media
All liquid cultures were grown in 2 X TY media which consisted of 16g
Bacto-Tryptone (Difco Laboratories), lOg Bacto-Tryptone yeast extract (Difco
Laboratories), and 5g NaCl per liter of water. Cultures were grown with shaking at
37°C.
(e) Plates, Agar, and Buffers
The composition of all plates, agar and buffers is given in Appendix I. All plates were either obtained from the Imperial College Media Service or kindly prepared by
Geraldine Davies.
(f) Enzymes
Klenow fragment—DNA Polymerase I was previously purified by Jian—Ping Shi or obtained from Boehringer Mannheim. T4 polynucleotide kinase and T4 DNA ligase were supplied by New England Biolabs, Inc. and Boehringer Mannheim respectively.
(g) Nucleoside triphosphates
All nucleoside triphosphates were purchased from Boehringer Mannheim.
Deoxyguanosine (dGTP), deoxyadenosine (dATP), deoxycytidine (dCTP), and deoxythymidine triphosphate (dTTP) were made up as lOmM stock solutions in 1 X
TE buffer and stored at — 20°C. A dNTP mixture refers to an equimolar concentration of all four deoxynucleoside triphosphates at the specified concentration. Radiolabelled
[7—^2p] ATP was purchased from Amersham International pic..
2.3.2B Mutagenesis Using the Kunkel Method
(a) Phage
The phage were prepard by the method in section 2.3.3A.
(b) Oligonucleotide
The sequence and melting temperature of the YTS17S18 oligonucleotide is listed in
Table 2.1. This oligonucleotide was synthesized and purified according to the procedures previously described in sections 2.1 and 2.2. 44
(c) Strains
Phage were grown on E. coii strain CJ236 (Table 2.2; Kunkel et al., 1987) for production of DNA template containing uracil substituted for thymidine. This strain was maintained on minimal plates supplemented with glucose (Appendix I) and chloramphenicol (10—30/xg/ml) which maintained selective pressure for production of the F —factor in this case. Transformation and plating cells were identical to section
2.3.2A.
(d) Other materials
The media, plates, agar, buffers, enzymes and nucleoside trisphosphates were the same as those used in section 2.3.2A. In addition the template preparation was enriched with uridine (Sigma Chemical Co. Ltd.).
2.3.3 Methods
2.3.3A Small Scale Preparation of Single—Stranded M13 Template for the Single
Primer and Amber Selection Approaches
1 to 2fil of a 1:1000 dilution of either M13mp93tyrTS or M13mp93AmIVtyrTS stock (10*0—10*2 pfu/mi) was streaked on fresh H plates using a sterile loop. A lawn of cells was provided by overlaying with 3ml of H top agar containing 200/d of an overnight culture of JM101 diluted in 2 X TY broth and grown for approximately 6hr at 37°C. The H plates were incubated at 37°C overnight.
Individual plaques from the overnight plates were toothpicked into 1.5ml of 2 X
TY broth containing a 1:100 dilution of an overnight JM101 culture. After growing 6hr at 37°C with shaking the cells were spun down in 1.5ml Eppendorf tubes using a microfuge at room temperature. The pellets were discarded after the supernatants had been transferred to fresh tubes.
150/xl of 50% polyethylene glycol—6000 (PEG)/2.5M NaCl was added to each
Eppendorf tube to precipitate phage. The tubes were quickly vortexed and left at room temperature for 5min. The tubes were then spun for another lOmin and the 45 supernatant was decanted. Remaining traces of PEG were aspirated with a glass Pasteur pipette. The tubes were briefly spun again and reaspirated. The pellet was then dissolved in llOjd of 1 X TE buffer.
Phage coat protein was extracted by adding 50 jd of phenol ’.chloroform (1:1, saturated in 1 X TE buffer) to each tube. The tubes were vortexed and left on ice for approximately 20min and then spun in a microfuge for 5—lOmin at room temperature. The lower organic phase was removed and the phenol ichloroform extraction was repeated. After separation of layers, 100/d of the top aqueous layer was removed and transferred to fresh tubes, each containing 10/d 5M NaCl.
The DNA was precipitated by adding 200/d of ethanol to each tube which was placed at — 20°C overnight. The following day tubes were spun in a microfuge at 4°C for lOmin to pellet the DNA. The ethanol was decanted and the tubes were allowed to dry.
The DNA pellets were dissolved in 100 /d of 1 X TE and the phenol .‘chloroform extraction was repeated once and followed by another ethanol precipitation overnight.
After pellets were spun and dried, 20/d of 1 X TE buffer were added to each tube which was then stored at — 20°C until needed. Typically, this scale of DNA preparation yielded 5/xg of DNA per Eppendorf tube.
2.3.3B Large Scale Preparation of Single—Stranded M13 Template for the Kunkel
Method (Kunkel et al., 1987)
Phage from an M13mp93tyrTS stock were diluted and streaked out on H plates as described in section 2.3.3A. All liquid cultures containing CJ236 were supplememted with chloramphenicol (30/zg/ml) to maintain the necessary selective pressure for Ml 3 infection.
Several individual plaques were toothpicked and grown in 5ml of 2 X TY broth containing 50/d of an overnight CJ236 culture and grown for approximately 2—3hr at
37°C with shaking. 100/d of this culture was innoculated into 200ml of 2 X TY broth containing 0.20g uridine. This culture was grown at 37°C overnight with shaking.
The culture was next split into two 100ml volumes then spun at 2,000rpm in a 46
Sorvall Centrifuge for 20min. The pellets were discarded and the supernatants were transferred to fresh centrifuge tubes. 10ml of 20% PEG/2.5M NaCl were added to each tube which was vortexed and spun again at 2,000rpm in the Sorvall at 4°C for another 20min.
After all traces of PEG were removed each pellet was redissolved in 2.0ml of 1
X TE buffer and was then transferred to 1.5ml Eppendorf tubes in 500/d aliquots.
Phage coat protein was extracted twice as before (section 2.3.3A) using 250/d volumes of phenol rchloroform. DNA was also precipitated using 50 jd 5M NaCl and 900/d ethanol per tube.
After DNA pellets were spun and dried the following day, 200jd of 1 X TE buffer was added to each tube. A final phenol :chloroform extraction followed by an ethanol precipitation was performed using volumes identical to section 2.3.3A.
DNA pellets were spun and dried again. 100/d of 1 X TE buffer was added to each tube which was then stored at — 20°C.
2.3.3C Kinasing Mutagenic and Selection Oligonucleotides
lOOpmol of each primer were phosphorylated at the 5* end in a reaction volume of 20/d containing ImM ATP, lOmM DTE (dithioerythritol) and 1 unit of T4 polynucleotide kinase in kinase buffer. The reaction was allowed to proceed for lh r at
37°C and then terminated by heating the samples to 65°C for 15min. The 5' end phosphorylated primers were stored at — 20°C.
2.3.3D Annealing, Extension, and Ligation
(a) Single Primer Approach
2/d (lOpmol) of 5' end phosphorylated mutagenic primer were incubated with 5/d (1/ig) of DNA template prepared in section 2.3.3A in 1 X TM buffer (total reaction volume, lOjd) at 80°C for lmin and allowed to cool slowly to room temperature over approximately lhr. Klenow (1—5 units) and T4 DNA ligase (1 unit) were added to the annealed mixtures with 0.5mM rATP, lOmM DTT, and 0.5mM dNTP's. The reaction was allowed to proc^d overnight in a water bath cooled to 15°C. If transformation 47 was not attempted the next day, reaction was stored at —20°C.
(b) Coupled Primer Approach With Amber Selection
The procedure was identical to above except that lOpmol of 5' end phosphorylated amber selection primer were added to the annealing mixture maintaining the same volume. The extension and ligation reaction with Klenow and T4 DNA ligase was as before.
(c) Kunkel Method
The procedure was identical to the single primer approach except that the uracil substituted template, prepared in section 2.3.3B, was used.
2.3.3E Preparation and Transformation of Competent Cells
200fd of an overnight culture of E. coli mutL (single priming or Kunkel Method) or HB2154 (amber selection) was grown for approximately 3—4hr at 37°C with shaking until reaching an A55Q = 0.3. The cells were harvested at 2,000rpm at room temperature for lOmin in a SS—34 rotor. After the supernatant was discarded the cells were gently resuspended in 5.0ml of cold 50mM CaClj and left on ice for approximately 20min. The cells were spun again as before, the supernatant discarded and the cells resuspended in 2.0ml of 50mM CaCl2. These 'competent' cells were typically left on ice for 24hr at 4°C before transformation was attempted.
lfd of the extension and ligation reaction mixture was added to 200/d of competent cells placed in a Sterilin tubes which was then heat shocked in a water bath at 45°C for 2min. 200/d of plating cells JM101 (single priming or Kunkel method) or HB2154 (amber selection) was added to the Sterilin and then added to
3.0ml H top agar which was poured on an H plate. The procedure was repeated for various amounts of the extension and ligation reaction mixture (2, 3, or 5jd aliquots for example) and all plates were incubated at 37°C overnight.
2.3.3F Phage Supernatant Screening By Hybridization With Mutagenic Oligonucleotides
Individual plaques were toothpicked from the overnight plates prepared in section 48 2.3.3E into 1,5ml of 2 X TY broth containing a 1:100 dilution of an overnight culture of JM101. The plaques were grown as described in section 2.3.3A, spun, and the supernatants transferred to fresh Eppendorf tubes.
A 1.0cm x 1.0cm labelled grid was drawn with a soft pencil on a nitrocellulose
(0.45/mi, 82mm diameter, Schleicher and Schuell) or nylon (Hybond, Amersham
International pic.) filter. 2—4jd of each phage supernatant (containing some single stranded Ml3) was spotted on to the grid. The DNA was attached to the nitrocellulose filter by baking it for 4hr at 80®C or in the case of the nylon filter, placing it on a
UV source for lOmin.
lOOpmol of the mutagenic olgonucleotide were 5' end labelled using 1 unit of T4 polynucleotide kinase and 50/iCi of [y—32p]ATP under the same conditions described in section 2.3.3C. Labelled primer was separated from unreacted ATP by collecting the eluant after spinning the reaction volume through a Sephadex G —25 column in a
1.0ml Sterilin syringe at 2,000rpm for 2min in a SS—34 rotor (Maniatis et al., 1982).
The filter was then sealed in a polythene bag with 2ml of hybridization buffer containing 100/d/ml of denatured DNA (e.g. salmon sperm, Sigma Chemical Co. Ltd.) and placed in a 60°C water bath for 2hr for the prehybridization step. Upon cooling the bag was carefully punctured and lOpmol of 5' end labelled mutagenic primer were added. The bag was carefully resealed and placed in the 60°C water bath which was then switched off leaving the filter to cool overnight.
The filter was removed from the bag the following morning and washed twice
(lmin per wash) at room temperature in 100ml of 6 X SSC buffer. The filter was dried on Whatman 3MM paper, wrapped in Saran wrap, and autoradiographed (using
Fuji X—ray Film, Medical grade, Fuji Photo Film Co. Ltd.) overnight at —80°C.
The next day the filter was washed in 100ml of 6 X SSC buffer at T^ — 4°C
(see section 2.3.1). The filter was blotted dry again, wrapped in Saran wrap and autoradiographed as before. Supernatants suspected of containing the desired mutation
'lit up' on the autoradiograph. If there was no discrimination, washes were repeated as necessary closer to and even above T^. The autoradiograph of a nylon filter displaying potential mutants is shown in Figure 2.2. 49
Figure 2.2 Phage supernatant screening by hybridization with a mutagenic oligonucleotide probe
The autoradiograph of a 54°C wash (T^ —2°C) to determine the presence of the
Thr51 — > Val mutation (2 base mismatch). In this example, there was already some discrimination between positive and negative signals at the less stringent room temperature wash (not shown). The effective removal of the probe was only achieved under the more stringent conditions pictured above. 50
2.3.3G Plaque Purification
Since transfection and plating were carried out without a round of replication and reinfection, a mixed population of phage could be present in the phage supernatants which remained hybridized to the filter after a stringent wash. Therefore, plaque purification was required before further characterization. However, as a result of the asymmetric nature of Ml 3 replication (based upon studies on the bacteriophage fl), most plaques (>80%) contain predominantly one type of phage (Enea et al., 1975).
After a round of plaque purification and rescreening, positives were sequenced to verify the presence of the mutation.
Supernatants identified as positives were restreaked onto H plates exactly as described in section 2.3.3A using JM101 as the lawn. Plaques were picked and screened by hybridization as section 2.3.3F. Positives were assumed to be >95% pure and were prepared for sequencing at this stage.
2.4 Dideoxy Sequencing of the tyrS gene
2.4.1 Introduction
DNA sequencing was carried out using the dideoxy method (Sanger et al., 1977) which utilizes chain terminating inhibitors following the protocol described by Bankier and Barrell (1983). Of special note was the use of deoxy—7—deazaguanosine triphosphate (deaza—dGTP) substituted for deoxyguanosine triphosphate (dGTP) in some of the sequencing reactions (Mizusawa et al., 1986).
Compressions in band spacing on DNA sequencing gels may occur when the sequence, particularly one which is G:C rich, at the end of a nascent chain can form a hairpin loop structure (Kramer and Mills, 1978). Such structures at the end of the
3' nascent chains during a sequencing reaction are relatively stable and may give rise to bands of anomalously high mobility during electrophoresis. To alleviate this problem dITP (deoxyinosine 5 '—triphosphate) can be substituted for dGTP in the sequencing termination and chase mixes (Mills and Kramer, 1979). In this case the inosine moiety, which lacks the amine group at position 2 in the guanine base, is only able to form 51 two hydrogen bonds when paired with a cytosine compared to the three hydrogen
bonds formed in a Watson—Crick G:C pair. It follows that I:C pairs are less likely to
form stable secondary structures and replacement of dGTP with dlTP in sequencing
reactions may resolve some compressions. However, there is a noticeable intensity loss
in the appearance of the bands in the I—track on autoradiographs of sequencing gels
and it is often necessary to run the same reaction using dGTP in parallel.
A slightly different view about the origin of compressions on sequencing gels
attributes this phenomenon specifically to aggregation or stacking of stretched G
residues through the formation of a Hoogstein base pair (Seela et al., 1982).
Deaza—GTP, which lacks the amine group at position 7 of the guanine base, can
destabilize a potential G:C Hoogstein base pair through the loss of one hydrogen bond.
The use of deaza—GTP in place of dGTP displays no noticeable intensity loss. In
addition, a comparative study which involved sequencing a DNA fragment having 85%
GC content revealed no difference in the ability of dlTP to resolve compressions in
comparison to dGTP. dlTP also produced false bands at other nucleotide positions in
the sequencing gel whereas none were observed using the deaza—GTP (Mizusawa et
al., 1986).
2.4.2 Materials
(a) Reagents
Nucleoside Triphosphates
All 2 '—deoxynucleoside triphosphates (dN) and 2 '3 '—dideoxynucleoside triphosphates
(ddN) were purchased from Boehringer Mannheim and made up as lOmM stock
solutions as previously described in section 2.3.2A.
Termination mixes (in 1 X TE buffer):
T mix 500mM ddTTP 6nW dTTP 122mM dCTP 122mM dGTP C mix 35mM ddCTP 122nN dTTP 6mM dCTP 122n*1 dGTP G mix lOOmM ddGTP 122mM dTTP 122mM dCTP 6n*l dGTP A mix 50mM ddATP 83nW dTTP 83mM dCTP 63n*l dGTP
The concentration of ddNTP's to obtain satisfactory chain termination was determined empirically from widely used conditions (Bankier and Barrell, 1983). 52
Deoxy—7 —deazaguanosine trisphosphate (deaza—dGTP) was purchased from Boehringer
Mannheim and prepared as a lOmM stock according to section 2.3.2A . Deaza—dGTP
was substituted for dGTP at the same concentrations in all mixes.
The chase was composed of a 0.5mM dNTP mixture.
(b) Enzyme
Klenow fragment (Boehringer Mannheim) was used at approximately 1 unit per clone.
(c) Gel
DNA sequencing reactions were routinely electrophoresed in a 20cm x 20cm x 0.035cm
7M urea/6% polyacrylamide gel, polymerized in the presence of 0.08% (v/v)
tetraethylmethyldiamine and 0.07% (w/v) ammonium persulfate.
(d) Buffers and Dyes
As listed in Appendix I.
2.4.3 Method
The Ml3 template (7ml prepared as in section 2.3.3A) was annealed to the
sequencing primer (amount used depended upon primer — determined empirically) in
10ml 1 X TM buffer in capped Eppendorf tubes by cooling from 80°C to 40°C
(approximately 30min).
The sequencing reactions were done in 1.5ml capless Sardstedt tubes by adding
2/d aliquots at the tops of the tubes followed by a brief spin in a microfuge. The sequencing reactions were started by adding 2/d of the Klenow to 2/d of annealed primer and 2/d of the corresponding termination mix. After 15min at 37°C the termination inhibitors were chased away by adding 2/d of prepared dNTP chase mix.
After a further 15min the reaction was terminated by the addition of 2/d of formamide dyes.
The DNA sequencing samples were boiled for 2min (to denature the DNA) and loaded immediately on to the polyacrylamide gel and electrophoresed at constant power
(37W) in 1 X TBE buffer. After electrophoresis the gels were fixed in 10% acetic acid, transferred to Whatman 3MM paper, dried on a gel dryer, and autoradiographed. 53
Figure 2.3 shows the autoradiograph of a DNA sequencing gel which displays the
presence of a site—directed mutation in the tyrS gene.
2.4.4 Summary of Sequencing Primers
Table 2.3 lists the set of primers used for sequencing the tyrS gene. The choice
of a primer for use in verifying the presence of a mutation was generally determined
by its distance downstream from the mutation and its reliabilty.
2.5 Results
Of the three mutagenic methods attempted success was achieved with the single priming and the coupled priming with amber selection approaches. The purification and kinetic characterization of the mutant proteins arising from these two methodsare detailed in the Chapter 3. With regard to the Kunkel method, preliminary results on the attempted insertion of a serine residue between positions 17 and 18 in the amino acid sequence indicated only the presence of a three base shift in the genetic sequence. The results of all three methods are given in Table 2.4.
Table 2.4 summarizes each method and mutation and also includes the observed mutation frequencies based on phage supernatant screening (section 2.3.3F). Admittedly, it is generally accepted that mutagenic successes are highly dependent upon the quality of the oligonucleotide and its design as well as the actual base changes attempted hence no real comparative study was made between the three techniques described here. However, the apparent mutation frequency observed using the Kunkel method of mutagenesis, 45%, compared well with the published protocol which claims to acheive frequencies of at least 50% (Kunkel et al., 1987). In addition, the relatively high mutation frequency acheived using the coupled priming approach with amber selection
(40%) as compared to the single priming approach (4%) is consistent with a true comparative study detailed by Carter (1985) on a mutation made in a similar region of the tyrS gene. In his study he used a transformation mix enriched for covalently closed circular DNA using an alkaline sucrose gradient which was said to improve mutagenic frequencies 2—fold. Since no such enrichment was attempted in this study it 54
Figure 2.3 Dideoxy sequencing to verify the presence of the Thr51 > Val mutation
A T C G A
In this autoradiograph of a DNA sequencing gel the base sequence is read from the anti —sense strand. The labels on the tracks shown are inverted for ease in reading the tyrS sequence. The sequence from the top of the gel marked A' to T' is:
5 '- ACTTGGCCGTC ATTTT - 31: clearly showing the ACC~>GTC base alteration coding for valine at position 51. 55
Table 2.3 Primers used in sequencing the tyrS gene
Primer Sequence (5 * — >3 * ) Position of codon at center of primer
Q1 CCCGCCTGCTCGAAGCG 60
Q2 AGCGTGCGCT CGCTTTT 86
Q3 AGT CGT AGTT GTTTTTG 124
Q4 AACT CGGT ACTT GAAAT 164
Q5 tccttttgccaataacct 207
Q6 TTTCTCTTTCTCCACCC 243
Q7 GCCTCACCAACCTCTTC 286
Q8 AAATT GGCAAT GTCGCC 329
Q9 TGGATGTCTTCGCGCGC 372
Q10 TTACTATCCCCACCGC Beyond 419
All primers were synthesized by Mr. Jack Knill—Jones with the exception of Q4 which is the Phe—> Seri 64 mutagenic primer and Q1 which is the SP1 primer (Carter, 1985) given by Dr. Hugh Jones and Paul Carter respectively. The primer Q10 was synthesized to be complementary to the region beyond the C—terminus of the tyrS gene using sequence data from Dr. G. Winter (Wells, 1987). 56 Table 2.4 Summary of results
m u tatio n Thr51->Val A la50->Pro Thrl7Aspl8-> Thrl7SerAspl8 method single priming coupled priming Kunkel method w ith amber s e le c t ion
s t r a i n s JM101 JM101 JM101 used BMH 71-18 mutL HB2151 BMH 71-18 mutL HB2154 CJ236
% mutant si 4% 40% 45% confirmation yes yes no^ by dideoxy seq u encing
iThe frequency of clones which hybridized strongly to the mutagenic primer after a stringent wash (Tq —4°C) in a phage supernatant hybridization screening (section
2.3.3F).
^Preliminary results indicated only the presence of a three base shift in the genetic sequence. 57 follows that the frequencies observed by Carter, 70% and 12.5% for the amber selection and single priming methods respectively, reflect a near doubling of the frequencies reported here. 58
CHAPTER 3
PURIFICATION AND KINETIC ANALYSIS
3.1 Aminoacyl—tRNA Synthetases
Aminoacyl—tRNA synthetases (reviewed by Schimmel, 1987) comprise a group of enzymes which esterify the twenty different amino acids found in the cytosol to their corresponding tRNA molecules in preparation for the translation step in protein biosynthesis. Zamecnik and coworkers determined that one of the roles of ATP in the protein biosynthesis pathway was to serve as a substrate for the enzymatic activation of amino acids prior to the formation of the peptide bond (Hoagland, 1955; Hoagland et al.,
1956). However, Berg (1956) was the first to purify and demonstrate the specificity of an enzyme (methionyl—tRNA synthetase) which catalyzed the activation of an amino acid
(methionine). Shortly thereafter it was discovered that the activated amino acid was bound to a 'soluble' (transfer) RNA molecule (Hoagland et al., 1957; Ogata and Nohara, 1957).
Berg and Ofengand (1958) then proposed an enzymic mechanism for linking activated amino acids to transfer—RNA molecules using tRNA synthetases. With the publication of the definitive paper describing the role of transfer—RNA molecules in the protein biosynthesis pathway (Hoagland et al., 1958) the true importance of these enzymes was soon realized.
3.2 The Expression and Purification of Tyrosyl—tRNA Synthetase
3.2.1 Introduction
Tyrosyl—tRNA synthetase (TyrTS) was first isolated and characterized from
Escherichia coli (Calendar and Berg, 1966ab). The enzyme was later isolated from Bacillus stearothermophilus (Koch, 1974). It is a dimeric protein containing 419 amino acids in each subunit, each having a molecular weight around 45,000. TyrTS from B. stearothermophilus has been cloned into Ml3 and subsequently sequenced (section 2.3.2A;
Winter et al., 1983). The protein is expressed as 20—50% of the total soluble protein in 59 Ml 3—infected host E. coli cells (Winter et al., 1982). This is due to the presence of a strong tyrS promoter and the high gene dosage per cell (100 —200 copies Ml 3 RF). The expression level of the TyrTS in E. coli was also improved 3—fold by deletions of the transcriptional terminator in the 5' non—coding region of the tyrS gene (Waye and
Winter, 1986).
The purification of wild—type and mutant tyrosyl—tRNA synthetases was based upon the original protocol of Koch (1974) using published (Wilkinson et al., 1983; Wilkinson,
1984) as well as unpublished modifications (John Akins, personal communication). The simplicity of all protocols is based upon the fact that TyrTS is a thermostable protein. A prolonged heat treatment at 56°C (section 3.2.2F) denatures most of the E. coli proteins present in the cell lysate. The remaining soluble material is separated using a typical
DEAE — ion exchange column followed by a final separation using either an FPLC Mono
Q column or a hydroxyapatite column.
3.2.2 Method
The general details of a typical purification scheme for a 2L preparation of cells are listed here to resolve any ambiguities. Phage stocks were obtained from standard stocks stored in Professor A.R. Fersht's laboratory or from preparations in section 2.3.3G for mutants constructed as part of this thesis. All strains and media are as described in section 2.3.2A. Plates, agar, and buffers are listed in Appendix I. All purifications were carried out by Mr. John Akins and/or myself.
3.2.2A Preparation of Phage Cultures
A colony of JM101 from a fresh minimal glucose plate was used to innoculate 5ml
2 X TY media and grown overnight at 37°C with shaking to saturation. The overnight culture was then diluted 1:100 in 2 X TY and grown as before for 4hr.
A loopful of three dilutions (1:100, 1:1000, 1:10000) of phage stock (1 0 ^ —10 ^ pfu//il) was streaked on individual H plates. 3ml of H Top agar containing 200/d of the
JM101 culture was poured on top and plates were incubated at 37°C overnight.
The following day plaques were toothpicked and grown in 1.5ml 2 X TY as for 60
template preparation (section 2.3.3A). After spinning in a microfuge for lOmin, phage
supernatants were decanted into 1.5ml Eppendorf tubes and stored at 4°C.
3.2.2B Verification of Overexpression
Before a phage stock is used to innoculate a preparative culture it is necessary to
verify that the TyrTS protein is being efficiently over—expressed.
Eppendorf tubes containing the cell pellets from section 3.2.2A were spun briefly in a
microfuge and all remaining traces of phage supernants were drawn off with a Pasteur
pipette under vacuum. The pellets were dissolved in 80/d of 1 X TE buffer containing
lOmM 2—mercaptoethanol (2—ME) and 0.1 mM phenylmethylsulfonyl fluoride (PMSF) and
then sonicated using a probe sonicator with a microtip (Heat Systems Ultrasonics) for
5 —10s at maximum intensity.
The tubes were spun for lOmin and the supernatants were transferred to new
Eppendorf tubes and placed in a water bath at 56°C for 30min. These tubes were then spun in microfuge for lOmin and the supernatants decanted and retained.
3.2.2C SDS Gels
Protein samples were routinely electrophoresed in 83mmx45mmxlmm 0.1% sodium dodecyl sulfate (SDS)/10% polyacrylamide protein gels (Laemmli, 1970), polymerized in the presence of 0.08% (v/v) tetraethylmethyldiamine and 0.07% (w/v) ammonium persulfate.
5 of each supernatant from section 3.2.2B and 5/d of protein sample buffer (Appendix
I) were placed in an Eppendorf tube and heated at 100°C for 2min. 7/J of this mixture containing approximately 1/ig of protein were loaded onto the gel. Proteins were electrophoresed at 40mA for 40min in 0.1M Tris—Bicine (pH 8.3), 0.1% SDS, and visualized by staining in 0.3% Coomassie Brilliant Blue B (Sigma Chemicals Co. Ltd.),
50% methanol, 10% glacial acetic acid, 40% water and destaining in 50% methanol, 10% glacial acetic acid, 40 water. Figure 3.1 shows a gel demonstrating good overexpression of the Thr51->Val mutant of the TyrTS enzyme. 61
Figure 3.1 Overexpression of the Thr51 — > Val TyrTS mutant
Lanes A—F display approximately 1 ftg aliquots from protein supernatants obtained from
* a standard mini — preparation of the TV51 mutant as described in section 3.2.2B and
electrophoresed on the SDS/polyacrylamide gel described in section 3.2.2C. Lane G
displays the pure wild —type TyrTS protein standard. In this example, mutant TyrTS is
being expressed sufficiently well in all lanes such that any corresponding phage supernatant could be used for innoculation of a culture for a preparative yield of
protein (section 3.2.2D). 62
3.2.2D Infection of Cultures for Preparative Yield of Protein
An overnight culture of JM101 was diluted 1:100 (total volume 10ml) and grown at
37°C with shaking for 4hr (O.D. s 0.3). lOO/il of a phage supernatant prepared in section
3.2.2B demonstrating overexpression of the TyrTS enzyme was then innoculated into the
10ml culture and allowed to grow for another hour. 2ml aliquots of this phage infected culture were next innoculated into four 2L flasks each containing 500ml of 2 X TY. The cultures were grown with shaking at 37°C overnight.
3.2.2E Cell Harvesting and Breakage
The following day cells were transferred to 500ml polycarbonate tubes and centrifuged in a Sorvall GS—3 rotor at 8000rpm, 4°C for 20min. The cell pellets were resuspended in
50ml (total volume) of 1 X TE buffer containing lOmM 2—ME and 0.1 mM PMSF, frozen in liquid nitrogen and stored at — 20°C until further use.
Prior to sonication the suspension was thawed in a 37°C water bath and frozen in liquid nitrogen. This was repeated once. Following a third and Final thaw the cell suspension was divided equally between six 40ml polyethylene tubes. These were packed in ice and each was sonicated using the medium probe for 4 X 30s at full power with 30s rests between bursts. The tubes were centrifuged in a Sorvall SS—34 rotor at 20000rpm for 25min at 4°C. The supernatants were decanted and stored at 4°C and the pellets were resuspended in 50ml (total) 1 X TE buffer containing lOmM 2—ME, 0.1 mM PMSF, and sonicated and centrifuged as before.
3.2.2F Heat Treatment and Ammonium Sulfate Fractionation
The supernatants from section 3.2.2E were pooled, placed in 56°C water bath and centrifuged using 40ml polyethylene tubes in a Sorvall SS—34 rotor for 25min at 4°C. The supernatants were then pooled and placed on ice.
The TyrTS was next fractionated by adding 0.226g solid ammonium sulfate (enzyme grade, BDH Ltd.) per ml of the heat treated supernatant and allowing it to remain on ice with stirring for 30min. This 0—40% precipitation was centrifuged as just described and the supernatants pooled. The TyrTS was pelleted in a 40—70% precipitation by adding an 63 extra 0.187g per ml of solid ammonium sulfate as before. This precipitation was centrifuged and the pellets were stored at — 20°C until further use.
3.2.2G DEAE—Sephacel Ion Exchange Chromatography
The ammonium sulfate pellets were resuspended in 15ml (total volume) 50mM potassium phosphate (KP) buffer (pH 6.5) containing lOmM 2 —ME, O.lmM PMSF, and dialysed against 2L 50mM KP buffer containing 20/xM sodium pyrophosphate, lOmM
2 —ME and O.lmM PMSF overnight at 4°C.
All steps of the column chromatography were performed at 4°C in KP buffer (pH
6.5) containing lOmM 2—ME and O.lmM PMSF. The dialysate with an additional 50ml
50mM KP buffer was applied to a 25ml DEAE—Sephacel ion exchange column pre—equilbrated with 50mM KP buffer at a flow rate of lml/min. A linear gradient of
450ml 50—300mM KP buffer was applied, and the TyrTS eluted at about 200mM KP buffer. The major peak and side fractions containing TyrTS (as determined by SDS PAGE and/or pyrophosphate exchange activity) were pooled and reduced in volume by membrane ultrafiltration (Amicon, Model 202; PM30 membrane, 62mm diameter).
3.2.2H Fast Protein Liquid Chromatography
Concentrated protein was dialyzed against 20mM Tris—HC1 (pH 7.5), 20mM 2—ME and O.lmM PMSF overnight at 4°C and spun in a microfuge for lOmin to remove precipitated protein.
The Pharmacia FPLC system described in section 2.2 was used. All steps of the column chromatography were performed at room temperature in Tris—HC1 buffer (pH 7.5) containing lOmM 2—ME and O.lmM PMSF. Prior to sample loading a Pharmacia MonoQ
HR 10/10 column (10mm x 10cm) was equilibrated with 20mM Tris—HC1 at a flow rate of lml/min. A portion of the dialysate, corresponding approximately to 3.0 A2go units, was applied directly to the column. A linear gradient (flow rate 2ml/min) of 25ml
0 —300mM NaCl in 20mM Tris—HC1 was used to elute the sample over 25min. TyrTS typically eluted at 225mM NaCl and the major peak and side fractions (assayed for pyrophosphate exchange activity) were separately dialysed against 144mM Tris—HC1 (pH
7.78) with 20/iM pyrophosphate present. Aliquots of the purified TyrTS were frozen in 64
liquid nitrogen and stored at — 70°C until further use.
A general criticism of this step of the purification is that the functionalization of the
column was no different to that used in the previous ion exchange step. The result was
that only a marginal increase in peak resolution was achieved due to the increased speed and concentrative effects of the FPLC system. As an alternative, hydroxyapatite column chromatography was utilized from which a higher purity of TyrTS, as judged by SDS
PAGE, was obtained.
3.2.21 Hydroxyapatite Column Chromatography
Hydroxyapatite (HA), crystalline Caio(P04)5(OH)2, is well known for its ability to bind substances which interact with calcium (particularly nucleic acids and phosphoproteins).
Such samples are bound to an HA column in a phosphate buffer of low ionic strength then selectively eluted by raising the phosphate concentration. Most proteins also bind to
HA columns and their elution is much the same. The mechanism of binding in this case, however, is not clearly understood. It is postulated that because the resolution of HA columns is so fine (as judged by gradient elutions of complex mixtures) binding may be sensitive to very slight changes in the configuration of proteins' surfaces (Freifelder, 1982).
Concentrated protein from section 3.2.2G was dialyzed against lOmM KP buffer (pH
7.0) containing lOmM 2—ME and O.lmM PMSF overnight at 4°C.
Prior to sample loading a 2.2cm x 30cm glass column (Wright Scientific Instruments) packed with 120ml Reactifs IBF (LKB) —HA Ultrogel (bead size 60—180 pm; available from
Life Sciences Laboratories Ltd.) was washed with 400ml 500mM KP buffer containing lOmM 2—ME and O.lmM PMSF at 4°C and then equilibrated with lOmM KP buffer under the same conditions. Approximately 30ml of dialysate corresponding to 75—100 A2gQ units were directly loaded onto the column using a flow rate of 0.5ml/min. The sample was eluted with an 800ml linear gradient from 10 to 200mM KP buffer (containing lOmM
2—ME and O.lmM PMSF). Using a flow rate of 1.3ml/min, the eluate was collected in
7.5ml fractions overnight at 4°C. TyrTS typically eluted in a peak range of 70—90mM KP buffer (verified by SDS gels). Fractions were then pooled and stored in 50% glycerol at
—20°C until further use. Protein purified in this manner cannot be flash frozen in liquid 65
nitrogen without a buffer change (i.e. Tris-HCl, pH 7.78).
3.2.2J Sample Purification
The purification schemes of a 3 X 2L preparative culture (section 3.2.2D) for two
TyrTS mutants, Thr51—>Ser (TS51) and Thr51—>Val (TV51), are shown for comparison
in Table 3.1. The column traces with accompanying SDS gels following the purification of
the TS51 mutant are shown in Figures 3.2a,b and 3.3a,b (courtesy of Mr. John Akins
who performed these steps of the purification).
3.3 Kinetic Characterization of TyrTS
Tyrosyl—tRNA synthetase (TyrTS) from B. stearothermophilus catalyzes the
aminoacylation of its cognate tRNA by the following two—step reaction (Fersht and Jakes,
1975):
(1) E + Tyr + ATP £ E*Tyr-AMP + PPj Eqn. 3.1
(2) E-Tyr-AMP + tRNATyr J E + Tyr-tRNATyr + AMP.
In solution this enzyme binds one mole of tyrosine per mole of enzyme dimer as
measured by equilibrium dialysis methods (Fersht, 1975: Bosshard et al., 1975).
Furthermore, TyrTS displays classic 'half—of—the—sites activity'. This was originally demonstrated by Fersht et al. (1975a) using ATP/pyrophosphate exchange kinetics (section
3.5) to show that the enzyme forms one mole of tyrosyl adenylate (step (1) of equation
3.1) per mole dimer (two active sites). Ward and Fersht (1988) have recently extended this observation by showing through kinetic studies using genetically engineered heterodimers (see section 1.2.3B) that the wild—type enzyme is asymmetrical in solution.
This result appears to disagree with the refined three-dimensional structure of the enzyme in which it appears as a symmetrical dimer (Bhat et al., 1982; Bhat and Blow, 1983). A more detailed examination of this apparent conflict is included in the discussion section at the end of Chapter 4 (section 4.7). The true rate and dissociation constants for the formation of the adenylate were determined using rapid mixing techniques (section 3.6;
Fersht et al., 1975b) in combination with the previously mentioned equilibrium dialysis 66
Table 3.1 Purification scheme of mutant TyrTS's
TS51 TV51 s t e p volume ^ s o 1 volume a 280 (ml) (ml)
4 0 - 7 0 % A S 2 51 660 52 940 precipi tat ion (dialyzed)
DEAE-Sephace 1 14 96 16 194
d i a l y s i s 106 88 101 201
HA-Ultrogel 8.6 37 16 132
l()ne A 280 unit = 0.719mg TyrTS
2AS ■* ammonium sulfate
In general, no A2go readings were taken before dialysis of the 40—70% ammonium sulfate
precipitation. Both preparations began from 3 X 2L preparative cultures (section 3.2.3D)
which were pooled before the heat treatment step (section 3.2.3F). In the examples above
the TS51 mutant yielded 4.4mg per liter culture (27mg total) of pure protein while the
TV51 mutant yielded 16mg per liter culture (95mg total). These yields are consistent with
the observations that mutants of the TyrTS which display enhanced catalytic activity for tyrosyl adenylate formation (i.e. TV51) appear to express more efficiently compared to those with decreased activity (i.e. TS51). 67
Figure 3.2 DEAE-Sephacel purification of the Thr51—>Ser TyrTS mutant
0 . 7 5 M
[KC1]
0.00N
Course of the elution (2.0ml/min)
a. The elution profile of the TS51 mutant from the DEAE—Sephacel column (see section
3.2.2G). Pooled peaks are labelled to correspond with the SDS gel shown below.
TyrTS A B C D TyrTS •Total A280' 52 44 33 66
: r
* * t' . ’ . . ___ j-I* l I ~~ _ * “ ~~ ‘ b. The SDS gel of the separation above. Total A2go' ^ ^ total A2go unit! In each pooled peak. Peaks A and B were selected and combined (A2g(p96; Table 3.1) for the next step of the purification scheme. 68
Figure 3.3 Hydroxyapatite purification of the Thr51—>Ser TyrTS mutant
1.5M
[KP]
0.1M
Course of the elution (0.5ml/min)
a. The elution profile of the TS51 mutant from the HA—Ultrogel column (see section
3.2.21). Pooled peaks are labelled to correspond with the SDS gel shown below.
TyrTS A B C TyrTS 'T o ta l A28 o '______5 ______7 37
b. The SDS gel of the separation above. Total Ajgo*a t*1® total Ajgo units in each pooled peak. Peak C is the purified TyrTS (A2gos 37; Table 3.1) used in subsequent kinetic and/or crystallographic studies. 69
method. In addition, ^ P —NMR studies showed that this step (1) of the overall reaction
proceeds through an associative in line displacement due to the nucleophilic attack of the
carboxylate group of the tyrosine on the a —phosphoryl group of ATP (Lowe and Tansley,
1984).
The charging reaction, step (2) of equation 3.1 has also been examined but in much
less detail. Early studies by Jakes and Fersht (1975) established that in the presence of
tRNA two moles of tyrosine bind per one mole dimer. Biphasic kinetics for the charging
reaction were also observed. In addition, protein engineering studies provided information
regarding the location and the binding mode of the tRNA on the TyrTS enzyme (see
section 1.2.3B; Waye et al., 1983; Bedouelle and Winter, 1986; Carter et al., 1986).
Current work suggests that the two moles of tyrosine bind sequentially to the same active
site during the aminoacylation of one mole of tRNA and that the binding energy released
in association with the second mole of tyrosine appears to increase the rate of the
dissociation of the Tyr—tRNA^y1" complex from the enzyme (Ward and Fersht,
unpublished results). The possible increased availability of pure B. stearothermophilus
tRNATyr from a synthetic gene (Borgford and Fersht, personal communication) makes the
development of pre—steady state assays for this step of the reaction seem likely.
3.4 Active Site Titration
3.4.1 Introduction
In the absence of tRNA^J^, tyrosyl adenylate accumulates on the reaction pathway
and forms a stable enzyme—bound intermediate (T |/2 = 4hr for the wild—type enzyme,
25°C, pH 7.78). Utilizing radiolabelled [^ C ]—tyrosine and inorganic pyrophophatase
E*Tyr—AMP complex can formed at an assumed stoicheometry of one mole of tyrosyl adenylate per mole dimer. This complex can be filtered and selectively retained on nitrocellulose Biters according to the procedure of Yarus and Berg (1970). Given the specific activity of the tyrosine the concentration of active enzyme is determined from subsequent scintillation counting. This technique is fairly insensitive to slight changes In the reaction conditions and in comparison with the more rigorous burst assay technique (Fersht 70
et. al, 1975a) yields similar results. The ability to determine precisely the amount of
active enzyme has been an essential factor in obtaining the accurate and reproducible rate
constants from the detailed kinetic studies of the TyrTS enzyme.
3.4.2 Method
A mixture (total volume 100/d) containing 144mM Tris—HCl (pH 7.78), lOmM
MgCl2, lOmM 2—ME, O.lmM PMSF, lU/ml yeast inorganic pyrophophatase (Sigma
Chemical Co. ltd.), 2mM ATP, 10/iM [^ C ]—tyrosine (500mCi/mmol; Amersham
International pic.) and a variable amount of TyrTS (from 0.01 to 0.5mg/ml) was prepared
and then incubated at 25°C for lOmin. Three 20/d aliquots of this mixture were spotted
onto nitrocellulose filter discs (Sartorius 100N, 0.45/un, 2.5cm diameter), pre—soaked in
144mM Tris—HCl buffer. Discs were then washed with 3ml of the same buffer under
suction and dried under a heat lamp. Total counts were determined by spotting 20fd of
the mix directly onto a disc and drying as before. Discs were counted in PPO/POPOP
scintillation fluid, 0.5% 2,5—diphenyloxazole, 0.03% 1,4—di—(2—(5—phenyloxazolyl))—
benzene (BDH Ltd.) in toluene using a Searle Analytic Inc. Delta 3000, 6891 liquid scintillation counter.
3.5 Pyrophosphate Exchange Kinetics
3.5.1 Introduction
The steady—state rate constant for the formation of tyrosyl adenylate can be determined by measuring the incorporation of radiolabelled pyrophosphate into ATP catalyzed by the enzyme (Calendar and Berg, 1966ab). Once tyrosyl adenylate is formed in the forward reaction, the pyrophosphate released can be exchanged with [32pj— pyrophosphate. The back reaction then forms [^2p]—ATP which adheres to activated charcoal and can be retained on glass fiber filters. The amount of radiolabelled ATP is determined by scintillation counting of the filters given the stoichiometry of the reaction and the specific activity of the pyrophosphate.
The initial rates of pyrophosphate exchange are determined from the slopes of counts 71 per minute versus time. Furthermore, plots of initial reaction velocity v versus v/[S] where
[S] is substrate concentration yield a linear plot (Eadie, 1942; Hofstee 1959), consistent with Michaelis—Menten kinetics. The y—intercept is Vm»* (initial reaction velocity at infinite substrate concentration) and the slope is Michaelis constant (the concentration of substrate at which v = iVmax).
3.5.2 Method
Approximately 0.1 /iM TyrTS was incubated at 25°C in 150/il 144mM Tris—HC1 (pH
7.78), lOmM MgCl2, 2mM 2 -M E , O.lmM PMSF, and 2mM [32P]-tetrasodium pyrophosphate (1 — 4cpm/pmol, Amersham International pic.). For ATP dependence concentrations of ATP varied from 10/iM to 20mM depending upon the AT^ of the mutant
TyrTS.
Seven aliquots of 20 fd were sampled at intervals which varied from 15s to 15min depending upon the ATP concentration under trial. Each aliquot was quenched in 0.4ml of a suspension containing 3.5% perchloric acid and 1% activated charcoal. The mixture was then placed on a glass fiber filter (Whatman GF/C, 2.5cm diameter) and washed under suction with 3 x 5ml lOmM sodium pyrophosphate (pH 2). The filter was dried by washing with ethanol and then counted in BBOT scintillation fluid, 4.5%
2,5—bis(5'—tert—butyl—benzoxazolyl—(2'))thiophene in 3:1 toluene :2—methoxyethanol.
Total counts were determined by spotting a quenched aliquot of the reaction mixture directly onto a filter and drying as before, lmin and 15min counts were collected using a liquid scintillation counter.
3.5.3 Results
The pyrophosphate exchange data and accompanying Eadie—Hofstee plot for the
TV51 mutant are shown in Figure 3.4. In the activation reaction TV51 displayed a 3—fold lowering of for ATP but no change in the value for compared to the wild—type enzyme. Figure 3.4 Pyrophosphate exchange data for the TyrTS TV51 mutant
[ATP] V v / [ATP] mM s ' 1 s " 1 mM”l
4 .0 7.3 1.8
2.0 6.6 3.3
1.0 5.2 5.2
0.5 4.1 8.2
0.25 3.5 14.0
0.125 2.0 16.0
Enzyme ^cat *M ^cat/^M s " 1 mM s -1M-1 w ild -ty p e 8.4 1.08 7800
TV51 8 .4 0.40 21000
The Eadie—Hofstee Plot for TV51
v/[ATP] 73 3.6 Pre—Steady State Formation and Pyrophosphorolysis of Enzyme-Bound Tyrosyl
Adenylate
3.6.1 Introduction
For many mutants of the TyrTS enzyme the pre—steady state formation of tyrosyl adenylate may be observed by monitoring a decrease in tryptophan fluorescence. The fluorescence decays exponentially, and the observed decay rate at saturating tyrosine concentration follows Michaelis—Menten kinetics when the concentration of ATP is allowed to vary. Rapid chemical quenching experiments established that this fluorecence change was indeed due to the formation of the enzyme.tyrosyl adenylate complex (E*Tyr—AMP; Fersht et al., 1975b). In this case, fccat is equal to £3 (the rate of tyrosyl adenylate formation) and Kyi is equal to Kz * (the dissociation constant of ATP) according to reaction Scheme
I (taken from Fersht, 1987):
Sch em e I
In addition, the E-Tyr—AMP complex may be isolated and the rate constant for the reverse reaction (to re-form the E-Tyr-ATP ternary complex) determined through varying the concentration of pyrophosphate and observing the increase in protein fluorescence.
With respect to Scheme I, the associated kcat is equal to k —3 (the rate of pyrophoroloysis) and is equal to *pp (the dissociation constant for pyrophosphate).
3.6.2 Method
The changes in the intrinsic protein tryptophan fluorescence were observed using a stopped—flow spectrophotometer (Fersht, 1975; Fersht et. al, 1975ab). This apparatus consisted of two driving syringes which delivered from 50 to 200 fjd of solution which was 74 then mixed and passed through a quartz cell for spectrophotometric observation. A third stopping syringe was placed behind the mixing chamber which, upon delivery of the solutions through the cell, hit a microswitch that triggered collection of the observed fluorescence change on a oscilloscope interfaced to the system. The experimental dead time was 1.7ms (Fersht et al., 1975a; Wells, 1987). The spectrofluorimeter consisted of a
150W xenon arc lamp, an f/3.5 grating monochromator, an EMI 9558 QB photomultiplier tube with a cut off filter at right angles to the path of excitation for monitoring emission, and an EMI 9781 A photomultiplier for monitoring excitation. Comparison of fluorescence to excitation was made using a Nicolet 3091 oscilloscope. This system was interfaced to a
BBC digital microcomputer (128K, Model B +) as described by Wells (1987). Time constants of the apparatus were set at least 10—fold smaller then the half-life of the reaction being monitored.
Protein tryptophan fluorescence was detected using excitation at 290nm and emission monitored at wavelengths greater than 330nm. The formation of the E-Tyr—AMP complex was accompanied by a decrease in fluorescence of approximately 6% following mixing of
0.2—0.3fcM enzyme in 144mM Tris—HC1 (pH 7.78), 100f*M tyrosine, and 1 U/ml inorganic pyrophosphatase (Sigma Chemical Co. ltd.) with an equal volume of 10/xM to lOmM MgATP (where [MgCy = [ATP] + 20mM) in 144mM Tris-HCl (pH 7.78) at a thermostatted 25°C (or 27°C, see section 3.7.3 for discussion). Data points were collected and fitted on-line to an exponential decay. The observed decay rate was then fitted to the Michaelis—Menten equation, k0\^ = [S]/(/£m + [S]) using a nonlinear least—squares curve fitting program, Enzfitter (written by Dr. R.J. Leatherbarrow) which gives a value for Jkcat and
Similarly, the pyrophorolysis of enzyme.tyrosyl adenylate complex was monitored by the increase in tryptophan fluorescence. The E-Tyr—AMP complex was first obtained by incubating 5—8pM enzyme with 2mM ATP and 10/iM tyrosine + [^^C]—tyrosine
(500mCi/mmol; Amersham International pic.) in 144mM Tris—HC1 (pH 7.78) at room temperature for lOmin. Excess ATP and tyrosine were removed using a Sephadex G —50 gel filtration column (1 x 10cm) equilibrated at 4°C in Tris buffer. The major peak was retained and a 20/d aliquot was spotted onto nitrocellulose and washed, dried and counted 75
as in an active site assay (section 3.4.2) to determine the concentration of the
E*Tyr—AMP complex in the collected fraction. Typically a solution containing 0.2—0.3/iM
E-Tyr—AMP and a lOmM excess of MgClj in 144mM Tris—HC1 buffer (pH 7.78) was
mixed in the stopped flow machine with an equal volume of 10 //M to 4mM tetrasodium
pyrophosphate at 25±0.1°C. The reaction was accompanied by an increase in fluorescence.
Collection and data analysis proceeded as previously described.
3.6.3 Results
The data for the formation of tyrosyl adenylate and pyrophosphorolysis in the TV51
mutant are shown with accompanying plots and Eadie—Hofstee transformations in Figures
3.5 and 3.6, respectively. The resulting rate and dissociation constants are given with those
observed for the wild—type TyrTS in Table 3.2.
The TV51 mutant displays a 4—fold increase in the rate of adenylate formation, k$,
and nearly a 24—fold increase in its affinity for ATP, ATa', compared to the wild—type
enzyme. These combine to give a 100—fold increase in the specificity constant, k^/K^'t
which is the largest value observed for any TyrTS mutant. The biological significance of
this result is dicussed in Chapter 5 (section 5.3). The accuracy of this data falls within
typical standard error ranges; ±5% for rate constants and ±10% for dissociation constants
(Wells and Fersht, 1986).
The data for pyrophosphorolysis reaction indicate that the TV51 mutant has decreased
the rate, k —3, 1.6—fold which is consistent with most other TyrTS mutants which display an enhanced activity for the formation of tyrosyl adenylate. However, because TV51 also displays an enhanced affinity for pyrophosphate (a 2.3—fold decrease in Kpp) its
pyrophophorolysis activity is enhanced nearly 1.4—fold. Other TyrTS mutants which demostrate enhanced activities for the forward reaction display decreased activities for the reverse reaction. In addition, the accuracy of this data falls outside typical standard error ranges; in this case the standard error is 11% for k — 3 and 32% for K pp Because of such inconsistencies and errors associated with this reaction it is advisable that pre—steady state data collection be repeated if possible. However, for comparison with the wild—type enzyme these values for A:—3 and *PP will be used to calculate the values for 76 Figure 3.5 Tyrosyl adenylate formation data for the TyrTS TV51 mutant
VarlAble Value Std. Err.
k cat 1.60663E+02 3.42034E+00 Km 1.95146E-01 1.62933E-02
Csubstrate] Rate Calculated Std. Devn.
t 3.OOOOOE-OI 1.17300E+02 1.15362E+02 9. 13000E+00 2.00000E+00 1.41600E+02 1.46382E+02 1.19700E+01 3 1. OOOOOE+OO t.40600E+02 1.34431E+02 1.33300E+01 4 2.30000E-01 8.74000E+01 9.02318E+01 2.19700E+01 3 1.OOOOOE-O1 5.76600E+01 3.44339E+01 1.08900E+01 6 3.C0000E-02 2.80000E+01 3.27693E+01 5.29000E+00 7 2.30000E-02 1.90500E+01 1.82433E+01 2.83000E+00 77
Figure 3.6 Pyrophosphorolysis data for the TyrTS TV51 mutant
— Vari abls Valu* Std. Err.
k cat 1.01333E+01 1.14881E+00 Km 2.73336E-01 8.83231E-02
Caubstrat*] Rat* Calculatad Std. Devn.
1 5.OOOOOE-Ol 0.75000E+00 6.34985E+00 1.22500E+0O 2 1.OOOOOE+OO 9.49000E+00 7.93674E+00 7.80000E-01 3 2.OOOOOE+OO 8.24000E+00 8.91410E+00 4.30000E—01 4 4.OOOOOE+00 9.04000E+00 9.48470E+00 1.37800E+00 3 3.OOOOOE—02 1.1OOOOE+OO 1.36593E+00 2.04000E-01 6 2.30000E-01 5.51000E+00 4.83871E+00 4.12000E-01 Table 3.2 Summary of rate and dissociation constants
fc3 Ka ’ *3 /* a* fc-3 ^PP ^-3/^pp enzyme (s-1 ) (mM) > ( s -1 ) (n*I) (s^ M ”1)
wi Id-type^- 38 4 .7 8090 16.6 0.61 27210
TV51 161 0.20 805000 10.1 0.27 37410
Table 3.3 Gibbs' free energies of complexes of the wild—type and TV51 TyrTS enzymes r-1 i o kcal s
complex wi Id-type^- TV51 TV51 temperature^ standard^ G G AC correction error
1 E-Tyr -6.71 -6.71 0.0
E•T y r•ATP -9.89 -11.76 >1.9
[E TyrA TP]* 5.41 2.69 2.7
E Tyr-AMPPPi -10.38 -13.40 3.0
E-Tyr-AMP -5.99 -8 .5 3 2.5
^Values for the wild-type enzyme are from Wells and Fersht (1986).
2 SilSltUcAA- l,'\ t }■ ^•Jr\jv' Was noY trfptrifiSwhlk — YaiuiZ ij fD hi as WlM- ^ p d 2A2;Vj/vie, fo/ cO^\p/lSdV\ 79 construction of the reaction profile and difference energy diagrams which follow.
3.7 The Reaction Profile
3.7.1 Calculation of Energy Levels
The activation of tyrosine by TyrTS is described by Scheme 1 (section 3.6.1). The coreesponding rate constants are defined: = * - t'* t = [E][Tyr]/[E*Tyr], Ka'= k _ a/*a'
= [E-Tyr][ATP]/[E*Tyr*ATP], etc.. The Gibbs’ free energy of each enzyme-bound complex was calculated from the measured rate and binding constants shown in Table 3.2 using the following thermodymanic equations (Wells and Fersht, 1986):
Gg - 0 (reference state) (1)
GETyr - RTln*t (2)
GETyrATP " RTln(JCtICa’) (3)
G [Tyr-AMP]* - RTln(kbT/h) - RTln(fc3/Ka *Kt ) (4)
GE Tyr-AMPPPi - -RTln(/c3//c_3V (5)
GE*Tyr-AMP " -RTln(fc3Kpp/fc_3Ka'Kt) (6) where R is the gas constant, T is the absolute temperature (298°K), fcb is the
Boltzmann's constant, and h is Planck's constant. The standard state is 1 M for ATP, tyrosine and pyrophosphate. The net contribution of the changes associated with a mutated residue which stabilize each enzyme—bound complex along the reaction pathway is the difference in Gibbs' free energy between each mutant enzyme complex and the corresponding complex in the wild—type TyrTS.
In the case of the TV51 mutant since the value of Kt was not determined the standard state was the E*Tyr complex (i.e. Gfe-Tyr = 0)* The TV51 mutant can be classified as an ATP—binding site mutant as residue 51 is located only 3.6A from the adenosine moiety of the tyrosyl adenylate in the active site of the enzyme and more than
8.0 A from the tyrosine substrate (see Figure 4.2a, section 4.1.2). Other mutations at position 51 have shown very small changes in the dissociation constants, for tyrosine (Ho and Fersht, 1986) and based on computer modelling studies TV51 is expected to behave in a similar manner. Furthermore, the practice of using the E*Tyr complex as the standard 80
state has already been shown to isolate the effects of the interaction on the binding of the
adenylate moiety and not to affect the relative energy levels of other states (Wells and
Fersht, 1986).
3.7.2 Discussion
The resulting energy differences for the formation of tyrosyl adenylate between TV51
and the wild—type TyrTS are shown in the form of a free energy profile in Figure 3.7a.
Figure 3.7b further illustrates the effects of the TV51 mutation on Gibbs' free energy in
the form of a difference energy diagram. The corresponding free energy values for both
figures are given in Table 3.3. TV51 appears to differentially lower the free energy of all
states of in which the enzyme is bound to the ATP moiety and may be classified as a
differential binding mutant according to the conventions of Albery and Knowles (1976). An
incremental increase in stabilization occurs through the production of the E*Tyr—AMP-PPi
complex. However, no decrease in stabilization between this complex and the E*Tyr—AMP
complex has ever been observed for a position 51 mutant which displayed enhanced activity for formation of tyrosyl adenylate. Noting this observation and the poor quality the pyrophosphorolysis data (section 3.6.3) the value for stabilization of tyrosyl adenylate on this mutant may not be a true indication of its actual behavior.
It is also worth considering how the aminoacylation reaction may be affected by the
TV51 mutation. Ho and Fersht (1986) pointed out that mutants with more stable tyrosyl adenylate complexes had lower turnover numbers with respect to the aminoacylation reaction (the charging of the cognate tRNA^'y1’; Fersht et al., 1985). The explanation given says that these mutants at position 51 bind tyrosyl adenylate too strongly and create a thermodynamic pit out of which the intermediates must climb. However, natural variants of the enzyme have evolved to bind the tyrosyl adenylate less strongly thereby avoiding this problem. Relative to the wild—type enzyme the TV51 mutant stabilizes the tyrosyl adenylate complex by 2.5kcal/mol (the largest observed for any TyiTS mutant). One would expect that TV51 would display the lowest turnover number of all mutants with respect to the aminoacylation reaction. However, the loss of binding energy between the
E*Tyr—AMP.PPi and the E-Tyr—AMP complexes (0.5kcal/mol; see Table 3.3) is a 81
Figure 3.7 Gibbs' free energies of enzyme—bound complexes
a. The Gibbs' free energy profile for the formation of tyrosyl adenylate and
pyrophosphate by the TV51 mutant and the wild—type TyrTS according to Scheme 1
(section 3.6.1) using standard states of 1 M for ATP, tyrosine and pyrophosphate. The
energy levels for the TV51 mutant are shown by heavy lines.
b. The differences in Gibbs' free energies between enzyme-bound complexes of the
TV51 mutant and those of the wild—type TyrTS.
E.=-o £ T Vo <\5> *cV 82 phenomenon similar to that observed with natural position 51 variants of the TyrTS only which bind the E*Tyr—AMP complex less strongly relative to the wild—type B. stearothermophilus enzyme (Ho and Fersht, 1986). How this destabilization might affect the expected rate of charging of tRNA^y1", particularly when the formation of tyrosyl adenylate is the rate—limiting, is a question which must merit further experimentation.
3.7.3 Uncertainites Due to Temperature Variation
As was mentioned in section 3.6.2 the data for the formation of tyrosyl adenylate was taken at 27°C(300K). The effect of this 2°C temperature change upon the value of the dissociation constant, ATa», can be estimated using the van't Hoff isochore:
dlntf/dT = AH(T)/RT 2
where T is temperature, R is the universal gas constant, and AH is the enthalpy of the reaction under examination. If one makes the assumption that neither AH or AS changes significantly with temperature, the above equation can then be written as the approximation:
lntf(T) * lntf(T*) - (AH(T*)/R}{1/T - 1/T*}
where T and T* are two different temperatures. For the wild—type enzyme it can be shown using the experimentally determined AH value for the formation of tyrosyl adenylate («12kcal/mol; Wells, 1987) that the difference in ATa» at 298K and 300K is insignificant. In addition, given the value of Ka% for the TV51 mutant at 300K, the value of AH needed to produce a 5% change (which is half of the standard error associated with the experimental technique) in this equilibrium constant at 298K would be approximately 38kcal/mol. This would mean that the difference in AH between the wild—type TyrTS and TV51 would be 26kcal/mol. Since there is only a 2kcal/mol difference in the Gibbs' free energy for this reaction, this large change in enthalpy would seem unlikely. Although there
may be more dramatic effects upon the rate constant for this reaction (£ 3 , also determined at 300K for TV51), for the purposes of discussion in the previous section and those that follow in Chapter 5 no account is made for the possible alteration of these values when determined at 298K. The author acknowledges that a rigorous analysis of the
catalytic properties of TV51 would demand a re—determination of Ka t and A: 3 at standard conditions. 83
CHAPTER 4
CRYSTAL STRUCTURE DETERMINATION AND REFINEMENT
4.1 Structural Studies of the Tyrosyl—tRNA Synthetase
4.1.1 Structure of the Wild—Type TyrTS
The tyrosyl—tRNA synthetase from Bacillus stearothermophilus was crystallized
from ammonium sulfate (Reid et al., 1973) to yield crystals suitable for data collection
of the space group P3j21 (a = b = 64.5X, c = 238.5X; a = (8 = 9CP, y = 120P)
which were initially used to collect data to 2.7X resolution (Irwin et al., 1976). The
enzyme was shown to be a symmetric dimer with one monomer in the crystal
asymmetric unit (Km = 3.04A/dalton). Initial protein engineering studies were based on
the crystal structure determined by Bhat et al. (1982) after the amino acid sequence of
the TyrTS became available (Winter et al., 1983). Subsequently, the structure was
partly refined to a crystallographic R—factor of 32% when all data was included to
2.1 X (Bhat and Blow, 1983) although the model contained only the first 319 amino
acids from the amino terminus. The final 100 amino acids (residues 320—419) were
disordered in these crystals and the density has remained uninterpretable to the present
day despite the numerous structural studies this enzyme has undergone.
The amino terminal structure, Figure 4.1, partially consists of an aJ@ domain
(residues 1—220) comprised of six strands (A, F, E, B, C, D), five in a parallel arrangement with only A antiparallel to the others. Between each strand is a least one and up to four (D to E) a —helices. The arrangement of strands B, C, and D and their adjoining antiparallel a —helices follow the topology of a Rossmann mononucleotide binding fold (Rossmann et al.t 1977; Bhat et al.y 1982). This domain is linked (221—246) to a second domain consisting of five a —helices (247—319), furthest removed from the dimer interface and not forming part of the active site. 84
Figure 4.1 The crystal structure of tyrosyl—tRNA synthetase
Schematic drawing of the existing amino terminal tertiary structure of the monomer unit of the TyrTS from B. stearothermophilus. The tyrosyl adenylate (see Figures 4.2a and b) appears to make polar interactions with sidechains from the helices spanning residues
47—57 and 164—182, and the strand of <3—sheet, B (from Bhat et al., 1982). 85
4.1.2 The Active Site
The residues constituting the active site of the TyiTS where the activation of
tyrosine occurs (section 3.3) were initially identified (Bhat et al., 1982) through
comparison with the earlier studies defining the structures of tyrosinyl adenylate, an
inhibitor of TyrTS, and L—tyrosine (Monteilhet and Blow, 1978); L—tyrosinol, an
amino alcohol derivative of L—tyrosine (Monteilhet et al., 1984); and the true
intermediate tyrosyl adenylate (Blow and Rubin, 1980; Rubin and Blow, 1981) bound to
crystals of the wild—type enzyme. Additional studies using a truncated mutant of
TyrTS (section 4.1.3) provided an improved set of interaction distances for the tyrosine
in its binding pocket (Brick and Blow, 1987).
Figure 4.2a shows the fully extended conformation of the tyrosyl adenylate in the
active site. The tyrosine moiety is bound in a slot at the bottom of a 10A deep cleft
adjacent to the central strands of the a/@ domain. The adenosine moiety lies in part
of a wide cup—like depression near strand B and the helix (47—57) linking it to
strand C. The adenine base is in contact with this helix as well as the continuation of
the F strand towards the carboxyl terminus. The ribose ring is located at the point
where strands B and E separate, near the carboxyl terminal edge of the 0—sheet. A
schematic representation of the tyrosyl adenylate bound to the active site residues is
shown in Figures 4.2b. Initial mutagenesis studies were carried out on residues,
implicated from early X—ray crystallographic work (see Blow and Brick, 1985),
identified as having probable polar interactions with the tyrosyl adenylate which could
affect catalysis and binding (denoted with a dotted line in these figures).
4.1.3 Truncated Mutant
As noted in section 4.1.1, residues 320 to 419 in the full length wild—type enzyme were disordered in the original electron density map. As a result refinement of the structure relied on a model which used calculated structure factors for the uninterpreted electron density. This estimation was reflected in the large crystallographic R —factor, 32%, and placed a computational limitation on the ability to fit the interpretable electron density. 86
Figure 4.2 The active site of TyrTS with bound tyrosyl adenylate
a. The fully extended conformation of the tyrosyl adenylate in the TyrTS active site,
showing the relationship between the various side chains and the substrate (taken from
Fersht et al., 1984).
His-48
NH, Gln-173-c;
Cys-35—sh
b. A schematic representation of the some of interactions between polar side chains of
TyrTS and tyrosyl adenylate that were implicated from X -ray crystallographic work (taken from Fersht, 1987; based upon Blow and Brick, 1985). 87
In an effort to gain more insight about the activity and structure of the ordered
region of the TyrTS, Waye et al. (1983) constructed a 'truncated' mutant which lacked
the disordered 100 residues from the carboxyl terminus. This deletion mutant was able
to catalyze the activation of tyrosine but lacked the ability to bind and hence charge
its cognate tRNA^y1". It crystallized in space group P2j with the molecular dimer
contained in the asymmetric unit (Vm = 2.73A/dalton). Its structure was determined to
2.5A resolution using molecular replacement techniques (Brick and Blow, 1987). The
refined model had a crystallographic R —factor of 18.7% and the model obtained from
this structure was used to further refine the original wild—type structure thereby
reducing its crystallographic J?—factor to 23% (P. Brick, personal communication).
The structure of the truncated mutant is essentially the same as the known
structure of the wild—type enzyme previously described (section 4.1.1). In the
truncated dimer, the cx/0 domains are related by an exact rotation of 180°; however,
one of the a —helical domains is rotated a further 4.7° from this 2—fold local
symmetry. In addition, tyrosine was present at high concentration (ImM) during
crystallization and good electron density for this substrate was observed in both subunits
of the dimer (see also Monteilhet and Blow, 1978). Solution studies (section 3.3) have
shown, though, that one molecule of tyrosine is bound per dimer; the conflict of these
observations is discussed at the end of this chapter (section 4.7). In the refined
structure of the truncated mutant the tyrosine substrate binds in a slot at the bottom
of the active site cleft in the a//3 domain. It is surrounded by polar side chains and
water molecules that are involved in an intricate hydrogen bonding network shown in
Figures 4.3a and b. Solvent structure, consisting of 143 water molecules, was also
assigned within the crystallographic asymmetric unit and henceforth served as a partial
model for the later refinement of the wild—type structure.
4.1.4 Model Building of the Transition State
31p—NMR studies on the reaction mechanism of the TyrTS have shown that the formation of tyrosyl adenylate involves an associative in line displacement due to the nucleophilic attack of the csrboxylate group of the tyrosine on the a —phosphoryl group 88
Figure 4.3 The tyrosine substrate in the binding pocket
ASP 78
THR73
ASNI23 ASNI23
ASP 176
a. A stereo view showing the structure of the tyrosine substrate in the binding pocket of
the truncated mutant (section 4.1.3). In this diagram hydrogen bonds are represented by
broken lines and water molecules are shown as double circles (taken from Brick and Blow,
1987).
Ser80 —n — H
b. A schematic representation of the hydrogen—bonding network around the tyrosine substrate binding pocket (taken from Brick and Blow, 1987). 89
of ATP. Magnesium pyrophosphate was eliminated in this reaction and an inversion of
the a —phosphoryl group was observed (Lowe and Tansley, 1984). It was postulated
that a pentacoordinate transition state of the a —phosphoryl group was necessary to
explain the stereochemical change. In addition, replacement of residue histidine 45 with
an asparagine caused a 2000—fold lowering of fccat whilst the values for ATP and
tyrosine were hardly affected (Fersht et al., 1984). His45, however, does not appear to
interact the with the tyrosyl adenylate in the crystal structure (see Figures 4.2a and b;
Fersht et al., 1984). Leatherbarrow et al. (1985) used this information with the crystal
structure of the enzyme—bound tyrosyl adenylate complex to model the active site with
a pentacoordinate transition state structure (shown in Figures 4.4a and b).
In this model the 7 —phosphoryl group of the intermediate could not only make a
hydrogen bond with His45 but also with residue Thr40. Subsequent mutagenesis of
Thr40-*Ala and His45-»Gly in combination with kinetic analyses indicated that the
binding energy contributed by these residues was primarily used in stabilizing the
transition state and the tyrosyl adenylate.PPj complex. Because no comparable effects were observed on the enzyme.substrate (ATP,tyrosine) or enzyme.tyrosyl adenylate complexes, the large stabilization of the transition state was interpreted to mean that residues Thr40 and His45 were responsible for a significant rate enhancement
(Leatherbarrow et al., 1985; Leatherbarrow and Fersht, 1987). Although no stable transition state analogue has been available for a crystallographic study of specific interactions such as those inferred here, this model serves as a useful starting point to base further hypotheses regarding transition state structure and theory.
4.2 Crystallization of TyrTS
4.2.1 Method
Crystallizations of mutant enzymes of the TyrTS were carried out under similar conditions to those of Reid et al. (1973). A typical protocol for most recent crystallization conditions is described below. All crystallizations, unless otherwise noted, were performed by Lesley Lloyd, John Akins (both of whom I thank), or myself. 90
Figure 4.4 The pentacoordinate transition state in the active site of TyrTS
a. The model of the transition state of tyrosine and ATP during the formation of tyrosyl adenylate based upon the extended conformation of tyrosyl adenylate observed in X—ray crystallographic studies (from Leatherbarrow et al., 1985).
Hls-45 A rfl-86^ Thr-40 Nil X ♦ A h C=- Nil. HN‘ ' * Nil, \ Lys-82^ __ ° w ° ‘ ♦ Lys-230 NH,. < / ' NH, | o / * Lys-233 Gln-173-c; 0» p ___ H»Nvy < / \ ♦ M 1 / i ' o
ElTyr-ATP]* b. A schematic representation of some the interactions between polar side chains of TyrTS and the transition state based in part upon modelling studies (His45 and Thr40; see section 4.1.4). Additional side chains shown (see Figure 4.2b for comparison) have been implicated from other mutagenesis experiments (reviewed by Fersht, 1987; picture taken from Fersht, 1987). 91
TyrTS was purified to homogeneity as described in section 3.2. After the hydroxyapatite purification step (section 3.2.21), protein was concentrated to
15—30mg/ml in lOOmM Tris/acetate (pH 7.3) using a Millipore centricon—30. 2ml of buffer were typically added to the concentrated protein and the centricon was centrifuged for lhr at 5000rpm 4°C in a Sorvall SS—34 rotor until protein sample volume was 100 —200/il. The procedure was repeated twice and after the final buffer change the concentration of the sample based on its absorbance at 280nm was determined (one A ^ q unit = 0.719mg/ml).
A crystallization solution containing lOOmM Tris/acetate (pH 7.3), 7mM
2—mercaptoethanol, lOmM MgCl2, 74% (v/v) ammonium sulfate, and saturating tyrosine (approximately ImM) was filtered through a 0.45 pan 'Swinnix' Millipore filter.
Equal volumes, from 10 to 25/d, of protein sample solution and crystallization solution were added together to a well in a siliconized depression slide for the "sitting drop" method of crystallization. 4—12 wells were prepared for a single crystallization attempt and the slide was then placed in a 25mm diameter Petri dish on a plastic grid.
25—50ml of 47% ammonium sulfate was added to the Petri dish which was then sealed with grease and a glass top and left to equilibrate at 15°C. If micro—seeding was attempted, the drops were left for 24hr then injected with ground hexagonal plates
(space group P3j21).
4.2.2 Summary of Mutants Crystallized
Mutant enzymes crystallized from 2 weeks to 2 months after the initial preparation as extremely regular flat hexagonal plates. Crystals have been grown for data collection with maximum dimensions of 0.50mm x 0.50mm x 0.80mm. Table 4.1 summarizes the specific crystallization conditions required for each mutant of the TyrTS and experimentally- determined space groups. Table 4.1 Crystallization summary
muta n t a 280 b u ffer^ H A S 2 sat m i c r o - space (per ml) PH d r o p / w e 11 Tyr s e e d e d g r o u p
TP51 14 7.0 37/47 yes no P 3 X 21 4.6 6.8 4 0 / 4 7 . 5 no no P 3 X 21
TA51 15 7.3 37/47 yes no P3^21
TG51 33 7.3 37/47 yes no P 3 X 21
TS51 16 7.3 37/47 yes no P 3 X 21
T V 5 1 12 7.3 37/47 yes no P 3 ! 2 1 3
H G48 12 7.3 37/47 yes yes P 3 X 21 R 3 2 4
A P 5 0 5 16 7.3 37/47 no yes P 3 X 21 R32
Ifinal buffer concentration - 50mM Tris/acetate
2AS - ammonium sulfate
3TV51 has only been crystallized and exhibits the hexagonal plate morphology of space group P3j21.
4Space group determined by Dr. Patrice de Meester.
^Crystal 1ized by Silvia Onesti. AP50 exhibits the crystal morphologies of the space groups listed. No crystallographic data has been collected yet.
oro<>£e.c 93
4.3 Data Collection
4.3.1 X -Ray Sources
The aim when choosing an X—ray source for the data collection of a protein crystal structure is to get a high intensity beam with an adequate wavelength to suit the diffracting power of the crystal without being strongly absorbed. The characteristic radiation emitted from copper (1.54 A ) and molybdenum (0.71 X) targets have proved to be technically convenient sources although the former appears to be more frequently used in many protein crystallography laboratories today. In both sealed beam tubes and rotating anode sources the X—rays are produced under vacuum from a beam of electrons accelerated through a voltage to strike a copper target. This converts some of the energy of these electrons into X—rays. The voltage is set as to enhance the emission of the CuKcq (1.541 A) and the CuKa2 (1.544A) characteristic line spectra.
Monochromatization of the beam is then achieved using a filter with the appropriate absorption edge (Ni in this case) or with a single crystal monochromator such as graphite.
Alternatively, data from protein crystals can be collected using X—radiation emitted from a synchrotron source. Synchrotron radiation provides a broad spectrum of electromagnetic radiation emitted by charged particles, typically electrons or positrons, moving at velocities close to the speed of light on a circular path. It is often preferred to traditional CuKa radiation because of its high brilliance (small source size, fine collimation and high flux, giving reduced collection time) as well as the availability of shorter wavelengths (reduced absorption errors and possibly increased sample lifetimes). At the synchrotron radiation source in Daresbury, U.K., electrons are first produced and accelerated in a preinjector (a linear accelerator or LINAC). They attain their final energy, 2GeV, in a booster synchrotron ring and are then injected into a storage ring. The storage ring contains an array of bending magnets which deflect the beam from a linear path thereby maintaining the desired trajectory. The power of the radiation is proportional to the circulating current of the electron beam, dependent upon the magnetic field strength which controls electron acceleration. 94
Subsequently the radiation emitted is a spectrum with a characteristic wavelength Xc inversely proportional to beam energy and magnetic field. Additional magnets such as multipole wigglers and undulators are placed into straight sections of the storage ring between bending magnets to futher modulate emission spectra to shorter wavelengths and to concentrate the electron beam. The beam is polarized and emitted tangential to the ring. The radiation is then filtered, monochromatized and focussed before it reaches the X—ray camera.
4.3.2 Photographic Methods
Large unit cells of radiation sensitive protein crystals have demanded data collection to be carried out for collection of the asymmetric unit in a given time, limiting as far as possible the exposure of the crystals to the incident beam. Screened photographic methods (precession and Weissenberg) collect a single layer of reciprocal lattice per film. Although such films are easily indexed, crystals are exposed for long periods of time because much data are blocked by the screen and must be recorded separately. Removal of the screen allows one to collect portions of many reciprocal lattice layers simultaneously and hence quickly. However, the complexity of the photographs makes indexing extremely difficult. With the advent of digitized microdensitometry and computer aided indexing, the technical constraints upon this method have been relaxed (Xuong et al., 1968; Arndt, 1968).
4.3.3 The Arndt—Wonacott Oscillation Camera (Enraf—Nonius)
In the screenless oscillation (rotation) method a three-dimensional data set is collected by taking a series of small angle rotation photographs. The total angle of rotation, p, for any given crystal required to collect a complete data set is primarily determined by the the crystal's space group, its orientation in the beam, and the desired resolution. The rotation angle for each photograph, A The basic requirements for the oscillation camera are only the rotation of the crystal spindle perpendicular to the incident X—ray beam and a flat or vee—shaped film to record the reflections. As the crystal is rotated individual reciprocal lattice points cut the Ewald sphere of reflection and are recorded on the film. This type of geometry gives rise to a blind region, a defined area of reciprocal space which cannot cut the reflection sphere. However, if data arc-collected using only one rotation axis, the amount of data lost is often negligible (3% of the total number of reflections at 3A resolution; Blundell and Johnson, 1976). Spots which come too close to the end of an oscillation range will only be partially recorded. The remaining portion is then recorded on the adjacent film. One of the main technical developments in the oscillation method is the ability of one to reliably sum the portions of such partially recorded spots. The precision of the spindle rotation in combination with its antibacklash features were shown by Arndt et al. (1973) to result in no more than a 0.005° difference in beginning of two adjacent films. Arndt et al. (1973) also claimed that the intensities of partially recorded spots summed between adjacent film packs showed no significant differences to the corresponding fully recorded reflections. In the data collection system used at Imperial College and the SRS at Daresbury (manufactured by Enraf—Nonius) incident beam from an X -ray source (CuKa or synchrotron radiation), first collimated to the desired cross-fire, strikes the crystal mounted on a spindle perpendicular to the beam. Optionally, the crystal may be cooled by a wide stream of dry air provided by a compressor equipped with drying towers and a cooling coil. A helium filled cone is placed between the crystal and the film holder to reduce air scatter. A lead backstop is fixed to a sheet of mylar on the back of the cone to stop the incident beam. The automated film change as well as the crystal rotation and exposure times are set using an electronic control box interfaced to the carousel, the spindle, and the shutter (a solenoid operated plunger located in the collimator). 96 4.4 Data Measurement 4.4.1 Densitometry Oscillation photographs were digitized on a Joyce—Loebl Scandig 3 rotating—drum microdensitometer controlled by either a Data General Nova 3/12 computer or a VAX—11/750 computer (Digital Equipment Corporation). All scanning was performed using a 50pm raster step size and an optical density range of 0.0 to 2.0 units. The program SCAN FILM then transferred digitized images from as many as three Elms scanned on the microdensitometer to a direct access file. The Hie contained optical densities in the range 0—255 stored one per byte. 4.4.2 Data Measurement Programs The digitized data were processed using a suite of programs originally described by Nyborg and Wonacott (1977) and subsequently extensively rewritten at Imperial College. The program procedures for typical data analysis are outlined here. To measure the intensity of fully and partially recorded reflections efficiently it is necessary to predict the diffraction pattern on each rotation film. In order to accomplish this the crystal orientation and unit cell parameters much be accurately known. Generally two photographs, stills, are taken at two crystal orientations 90° apart. Once these films are scanned the program STILLS selects a set of strong spots in each photograph and writes out their coordinates to a file. The refinement of the crystal cell orientation and unit cell parameters (including the unit cell size and angles, the crystal to film distance, the mosaicity, and if necessary the wavelength) proceeds using the program IDXREF. The prerequisites at this stage are only that the unit cell dimensions are known within 1%, the misorientation angles within 1°, and the position of the film in relation to the X—ray beam and the horizontal plane is established (using fiducial marks and if possible, a direct beam spot). The program then calculates fractional Miller indices from the input coordinate file created by STILLS. After rounding to the nearest integers, these indices together with the corresponding fractions and rotation angles are used for the refinement process (see Schwager et al., 1975; 97 Jones et al., 1977). Once crystal orientation and unit cell parameters are accurately known a file containing a list of reflections with the calculated positions and partialities can be generated for each oscillation film using the program OSCGEN. The integrated intensities of reflections on each film can then be measured using the program MOSFLM. As in IDXREF the position of the film in relation to the beam is established first. An initial search for reflections confined to the center of the film is made using the calculated diffraction pattern. Once a satisfactory agreement is achieved the remainder of the film is refined to fit the calculated pattern allowing for various film distortions. Optical densities from individual spots are then collected. The final output file contains the integrated and/or profile fitted intensities, positions, partialities, and standard deviations for all measured diffraction spots. A post—refinement of crystal orientation and cell dimensions from measured partial spot data can be carried out using the program POSTCHK. For any two adjacent contiguous films the consistency between the calculated fraction of a partially recorded reflection and the observed fraction is compared and an output is created containing the agreement statistics. Furthermore, a file can be created containing the indices and observed partialities which can then serve as an input file for the program IDXREF. The cell dimensions can then be more accurately refined and changes in crystal mosaicity (or beam divergence) and orientation (indicative of slippage) identified. 4.4.3 Data Scaling Programs Prior to calculation of the difference Fourier for each mutant structure all optical density data must be appropriately scaled and sorted. The program ABSCALE reads the rotation film data produced as a file from the program MOSFLM (preceding section 4.4.2). It first applies oblique incidence film absorption corrections for the flat (or vee—shaped) film geometry (Arndt and Wonacott, 1977) and then calculates scale factors between films in a pack. The scale factors are based on a comparison of intensities between reflections on all films in a pack using the Fox and Holmes (1966) 98 algorithm. It proceeds to apply Lorentz—polarization corrections and writes the fully scaled and corrected data to an output LCF file. The averaged scaled measurements and deviations from the mean are output in the form of a histogram based on both observed intensities and resolution. Two R —factors are calculated: one for the overall fit of each film in a pack, R/^q \ and f^SYM which compares the agreement between fully recorded symmetry related reflections in a pack. In both R —factor calculations *AB and f?SYM are derived from the following equation: N — E E 11(h) - 1(h) R - h 1-1______Eqn. 4.1 N £ E I(h)i h i-1 where I(h)j is the ith measurement of reflection h, and 1(h) is the mean value for N equivalent reflections. The intensities from fully recorded reflection on all films are then sorted on their Miller indices (after these have been transformed into a unique region of reciprocal space) and merged into a single file. Scale factors and temperature factors for each pack are then calculated according to the Fox and Holmes (1966) algorithm using the program ROTAVATA. The agreement of individual packs within the entire dataset is provided so that one may selectively remove packs which deviate too far from the mean before calculation of structure factors. The scale factors calculated by ROTAVATA are next applied to the entire data, partially recorded reflections summed, and a file containing final averaged intensities with accompanying temperature factors produced by the program AGROVATA. Poor agreements between common reflections are also rejected by this program and a final statistical profile is produced. This contains data distributions as a function of resolution and a comparative analysis of fully versus partially recorded reflections including standard deviations and fractional deviations from the mean. Finally, the averaged intensity data are converted to structure factor amplitudes using the program TRUNCATE. Data can be optionally truncated using French and Wilson's procedure (1978) which relies on the application of Bayesian statistics to Wilson's distribution for centric and acentric reflections. Better values are thus obtained 99 for structure factors calculated from weak data which would otherwise follow the the relationship IF* = A. 4.4.4 Data Collection and Processing Statistics The data collection and processing statistics for the five mutants whose refined structure is presented as part of this thesis (section 4.6.3) is shown in Table 4.2. All crystals used were isomorphous with the wild—type TyrTS (space group P3|21; a = b = 64.5X, c = 238.5X; ot = 0 = 9CP, 7 = 12(P). 4.5 Difference Electron Density Map Calculation 4.5.1 Theory The use of difference Fourier synthesis to study the structural changes in proteins arising from specific alterations to certain residues dates back to original chemical modifications of hemoglobin (Moffat, 1971) and lysozyme (Blake, 1967). It is the primary technique used for the determination of crystal structures of proteins whose residues have been altered using site-directed mutagenesis techniques (see Chapter 1). The success of the difference Fourier technique is based on the assumption that small structural changes within a large protein molecule will give rise to structure factors with nearly the same phase as the original 'wild-type' molecule. The coefficients of the Fourier synthesis become (Stryer et al., 1964): ( 't W 1 - 1 FcaZ1 )exP['°!ca/] 4 2 where I F0^51 and I Fca[ 1 are measured structure amplitudes of two similar noncentrosymmetric structures, in this case the wild—type ( cat) and the mutated (obs) protein structures; acai is the phase of Fca\. The Proof for the acentric case is shown in the form of an Argand diagram in Figure 4.5. The quality of an electron density map calculated in this manner depends upon (i) the quality of the wild-type protein phases, a; (ii) the errors associated in intensity measurements; and (iii) the 100 Table 4.2 Summary of data collection Mut ant T P 5 1 TP51 TA51 TG51 TS51 HG48 (no tyr) 0 W a v e length (A) 1 . 5 4 1 8 1 .488 1.506 1.488 1.488 1.488 S o u r c e C u K a syn* s yn syn s y n syn Crystal-to-film 80.1 83.4 84.7 82.5 82.5 82.5 distance(mm)^ Exposure time 3 . 0 x l 0 4 50 50 75 75 75 (sec/degree) Temperature(°C) -10 -13 -10 -10 -10 -10 C o l l e c t e d 2.5 2.5 2.6 2.5 2.5 2.8 o Resolut ion(A) A c c e p t e d 2.5 2.5 2.8 2.5 2.5 2.6 Resolut ion(A) N u m b e r of 2 1 1 1 1 1 c r y s t a l s N u m b e r of 15 16 15 14 14 14 f i l m packs Osc. angle from - 2 .5;72.5 180 . 0 95.0 186.0 8 0.0 -16.0 to 7 . 5 ; 9 2 . 5 220.0 132.5 221.0 115 . 0 19.0 increment(deg.) 2.5 2.5 2.5 2.5 2.5 2.5 N u m b e r of 4 0 9 4 8 4 8 8 4 7 436 1 9 42571 4 3 0 0 0 428 1 0 measurements^ Number of ind. 1 9 6 7 4 20067 19029 19872 20008 20145 reflect ions^ Merging R-factor 4 to col. res. 8 .8% 6.5% 12.9% 7.2% 9 . 9 % 8 .1% n to acc. res. n 10.7% n tl 7.6% *syn = Daresbury Synchrotron Radiation Source, Station PX 7.2 ^These values represent refined crystal—to—film distances determined within the program IDXREF (section 4.4.2). ^For collected resolution. ^The merging R—factor is defined as I(I(i) — < /> )/!/(« ), where 7(i) are the intensity values of the individual measurements, and < 7> the corresponding mean values (from Brick and Blow, 1987). 101 approximation itself. For a complete discussion about the effects of these errors and their treatment see Henderson and Moffat (1971). Interpretation of the significant features arising from the difference Fourier synthesis is dependent upon the levels and patterns of the various peaks present on the calculated difference electron density map. Given the fact that proteins are non—centrosymmetric structures equation 4.2 can be expanded using the cosine rule as follows (taken from Blundell and Johnson, 1976): f^ _j. Fca /fcos(0 - a) Fobs ~ Fcal Fobs ^ Fca l FoZ>s Fca Z But cos(0 - a) Hexp{i(0 - a)} + exp{-i(0 - a)}] and therefore (Fobs " Fca/)exPio “ f GXP— --- Fobs + FcaZ + FcaZfexP10 (ii) F o b s F caZ 4. FcaZfexP!<“* + 2a> ( H i ) Fobs + F caZ where a = acai and f and 6 represent the structure factor and phase of the difference structure as depicted in Figure 4.5. Term (i) represents the image of the protein structure but since F cai & F0fo and both F cai and F0bs > > ^ contribution is small. Term (ii) gives rise to the transform of f/2 in the electron density difference map. Term (iii) gives rise to noise. This reduction of peak heights by approximately half in this acentric Fourier synthesis was first analyzed by Luzatti (1953) for the case when a relatively small number of atoms are not included in the phase determination. To bring the scale of densities closer to their true values a factor of 2 may be used to weight the original coefficients. 4.5.2 Programs The calculation of the various difference Fouriers was made using a selected set of programs adapted to run on the VAX 11/750 computer at Imperial College. The first step in the calculation of the difference electron density map was to generate a 102 Figure 4.5 Construction of the isomorphous difference approximation for the acentric case An Argand diagram showing the relationship between the wild—type (F) and the mutant (Ffj) structure factors. The structure factor f represents the difference structure (taken from Henderson and Moffat, 1971). 103 set of structure factors for the wild—type protein (i.e. Fca/). This is done using the program GENSFC which, given the set of atomic coordinates from the refined wild—type structure, computes the corresponding electron density distribution. A space group specific reverse Fast Fourier Transform is applied to this density and yields a set of calculated structure factors. The structure factors, ^ 0bs an<* ^cal, are scaled together after a statistical analysis which assesses the quality of the data and deletes bad data on the basis of calculated standard deviation from the mean. The difference Fourier density is then calculated in crystallographic coordinates using the program FFT (Ten Eyck, 1973). The output density is interpolated to a standard Cartesian frame using SKWPLANES (Bricogne, 1976). If desired the 'skewed* map can be converted into a format suitable for use in the display and modelling program FRODO (Jones, 1978). 4.5.3 Results The electron density maps showing the difference in density between each of five different TyrTS mutants and the wild—type enzyme are shown in Figures 4.6a—e. All maps are superimposed on the structure of the wild—type TyrTS. Residues 48 to 51 are labelled in all figures for orientation within a segment of the structure comprising residues 45 to 55 (45/55). The contour levels for each map were chosen to display the most significant features for each individual structure where a is the root mean square density throughout the unit cell. All maps were obtained using Fourier coefficients (Fmut “ Fwild—type) an<* phases calculated from the refined wild—type structure including the well-ordered solvent molecules. Figure 4.6a displays the difference density close to residue Thr51 in the TP51 mutant (without tyrosine in the active site). The strongest features represent the substitution of proline for threonine. Specifically, positive density is observed at the C7 and C6 positions for proline, with an equal amount of negative density close to the positions of O7I and C72 of the original threonine residue. The negative and positive density above and to the right of Thr51 suggest a localized rearrangement of solvent; however, these features disappeared in the subsequent refinement of this structure scut T q>^ EmcIC^i .T. AiS. 104 Figure 4.6 The Fmut~ F\\Ud-tyrc difference election density maps of TyrTS mutants superimposed on the wild-type structure (see text for details) a. Thr51 — > Fro (TP51) mutant; 2.5X resolution; contours at +4.6(7 (blue) and — 4.6u (red). b. Thr51->G ly (TG51) mutant; 2.5A resolution; contours at +4.1(7 (blue) and — 4.1(7 (red). 105 Figure 4.6 (continued) c. Thr51 —> Ala (TA51) mutant; 2.8X resolution; contours at + 3.3tr (blue) and — 3.3a (red). O e. His48 — > Gly (HG48) mutant; 2.6A resolution; contours at +3.2a_ (blue) and — 3.2cr (red). 106 Figure 4.6 (continued) d. Thr51 — > Ser (TS51) mutant; 2.5A resolution; contours at + 3.2cr (blue) and -3.2a (red). 107 (section 4.6.3). No other difference density was observed around the 45/55 segment which might have indicated mainchain movement propagating from the introduction of the proline residue, even when contouring at levels near noise (±1 a). In constrast, complete removal of the threonine side chain in the TG51 mutant (threonine replaced by glycine) resulted in a series of small mainchain movements which appeared to extend from the carbonyl group of residue 51 through and possibly beyond the carbonyl group of residue 49. This is shown in Figure 4.6b by the equally distributed negative and positive peaks surrounding the mainchain in this region. The negative density representing the presence of the mutation is located near the O7I and C72 positions and appears to extend beyond the C01 position of the threonine. Both difference electron density maps shown in Figures 4.6a and b are shown at approximately the same contour levels to demonstrate the obvious differences between the presence and absence of localized mainchain movement. In Figures 4.6c and d, a 90° rotation of the previous figures to yields a view from the top of the 45/55 segment shown here to begin with residue 48. As already seen in the case of the TP51 mutant substitution of Thr51 with an alanine, mutant TA51, also leads to effectively no related mainchain movement in this portion of the structure. The strongest feature on this map, Figure 4.6c, is only the expected negative density near the O7I and C72 positions of Thr51. The peaks of negative and positive density located to the left of position 51 in this map appear in the portion of the active site where the adenylate moiety of the tyrosyl adenylate would be located (see Figures 4.2a and b). This 'disturbance' appears in roughly the same place for all mutants which contain tyrosine in their crystal structures (TA51, TG51, TS51 and HG48). Because the wild—type structure does not contain tyrosine these peaks appear to be related to the arrangement of solvent in the ligand—bound and ligand—free forms of the enzyme. Addition of tyrosine in the later refinement of these structures causes the majority of these features to disappear. Figure 4.6d shows the difference map of the TS51 mutant (serine replacement) in a similar orientation to the TA51 mutant shown in Figure 4.6c for a comparison of the isolated absence of the C72 atom evidenced by the large negative peak located 108 near this position. At higher contour levels (>4o) the small features seen within the 45/55 segment in this map disappear and do not represent any significant mainchain movement (as shown by subsequent refinement of the structure). Located to the left of position is the previously described peak 'disturbance' in the unoccupied portion of the active site. Figure 4.6e shows the difference electron density map of the HG48 (His48—Gly) mutant. A strong negative peak surrounds a large portion of the histidine side chain including the imadazole group confirming the presence of the mutation. In addition, the complete removal of this group also results in a propagated movement along the mainchain extending from residue 48 through residue 51 as was seen earlier with the TG51 mutant (Figure 4.6b). In this case a greater proportion of the mainchain and sidechain residues appear to be affected although the magnitude of the movements cannot be accurately compared without refinement. It is interesting to note that complete removal of a side chain at either end of this group of residues appears to disturb the same set of residues. 4.6 Refinement 4.6.1 Hendrickson—Konnert Restrained Least—Squares Refinement The refinement of the mutant structures presented as part of this thesis used the the restrained least—squares algorithm of Hendrickson and Konnert (1980). The input file for this refinement is prepared using the program PROTIN which compares the observed protein geometry with 'ideal' geometry. The refinement is then carried out using the program PROLSQ. The aim of this program is to minimize the difference between the observed and the calculated structure factors (F0^5 and FCflj, respectively) by supplementing the observed structure factor amplitudes with information about stereochemical restraints. Parameters to which restraints are applied include bond lengths and angles, torsion angles, planarity, chirality, and van der Waals' contact distances. The parameters minimized can be weighted between X—ray based and geometry based refinement. Once a weight is chosen with respect to a particular \ 'VC-'f Qy (AiSC-MSSi (jn £-(- H h S Z SjZ. W .i \ H laacUj, 4c\f'c) COWpAtzA C o o w it/ M. lY' 109 resolution range the structure factors and their derivatives are calculated using FFT algorithms (Ten Eyck, 1973). Applying a least—squares minimization, the conjugate gradient procedure, the parameter shifts are computed. The procedure is iterated until a convergence is achieved. The program then prints a statistical profile of the various parameters with their accompanying shifts and the overall R —factor for the average Fobs - Fcai discrepancy. 4.6.2 Method Because of the similarites regarding refinement of all five mutant structures, the general procedure followed by myself and Dr. Patrice de Meester (whom I thank) is outlined here. After examination of the initial Fobs — Fcai map (section 4.5) the coordinates of each mutated residue were modelled and, when appropriate, the tyrosine substrate coordinates were added using the program FRODO (Jones, 1978). Five to ten cycles of refinement of the entire structure on a VAX 11/750 computer (Digital Equipment Corp.) using PROLSQ were allowed to proceed until convergence was reached. Another set of difference maps was then calculated between the observed mutant structure factors and those derived from the newly refined model. Statistically significant peaks remaining on the Fobs — FCfl/ map were identified and examined using the programs ATPEAK and PEAKMAX (T. Skarzynski, personal communication). A 2Fobs — FCfl/ map was then calculated and used in conjunction with the model building capabilities of FRODO on an Evans and Sutherland PS300 graphics system to adjust mainchain and side chain coordinates as necessary. Water molecules with difference density at least three standard deviations above the zero level of the Fobs — Fcai map were added or deleted accordingly. Refinement continued in this manner, with additional model building if necessary, until a final convergence was reached. 4.6.3 Results Good geometry and low crystallographic R —factors were obtained from the least—squares refinement procedure described above. A summary of the deviations of 110 each mutant structure from ideal geometry and final R—factors are listed in Table 4.3. The coordinates for new side chains, water molecules or substrates (e.g. tyrosine) are listed in Appendix n. Appendix III contains two-dimensional plots which show changes in the overall separation of atoms and associated temperature factors for each mutant structure when compared with the wild—type TyrTS structure. A difference distance map which depicts shifts in a —carbon distances for each mutant structure is also included in this appendix (a description precedes these figures). The refined structures of residues 46 —54 in the wild—type and mutant TyrTS's are shown in Figures 4.7a—e and g. In these drawings mainchain and side chain atoms not altered during mutagenesis are shown in black. Water molecules are shown by two concentric black circles. The new side chain in each mutant structure is shown in red and the coordinates of the adenosine moiety of the tyrosinyl adenylate in the wild—type complex (Monteilhet and Blow, 1978; section 4.1.2) are shown in blue. The temperature factors of the mainchain atoms and the r.m.s. differences in A between mainchain and common side chain atoms of wild—type and each mutant for the 45/55 segment are given in Tables 4.4 and 4.5, respectively. As anticipated from the ^mut ~ ^wild—type electron density difference map (Figure 4.6a), no significant structural alterations occurred in the TP51 mutant. With respect to the 45/55 segment, no coordinates shifted by more than 0.2A and the temperature factors all followed the same general pattern and magnitudes displayed by the wild—type structure (see Tables 4.4 and 4.5, respectively). The TP51 coordinates also include seven new water molecules with temperature factors ranging from 19.9 to 34.1 A^. Only one new solvent molecule, HOH 601, appeared near the mutational site. This water molecule is within hydrogen—bonding distance of the carbonyl oxygen of Gly47 and would be displaced upon binding of the tyrosyl adenylate because of its proximity to the adenosine moiety as shown in Figure 4.6b. A water molecule with similar coordinates named HOH 601 (see Appendix n) also appears in the TG51 and TS51 mutant structures with a nearly identical temperature factor, approximately 32 A^, indicative of partial disorder or occupancy. Brown et al. (1987) also described the appearance of a new water molecule in the vicinity of the Thr51 residue in the Table 4.3 Deviations from ideal geometry after refinement and final R — factors Mutant TP51 TA51 T G 5 1 TS51 HG48 (no tyr) Input a Root - m e a n - square deviat ion O Distances (A) Bonds(1-2 0.0251 0.011 0. 0 1 4 0.012 0.012 0 . 0 0 7 nei g h b o r ) Angles(l-3 0.04 0.0 4 0 0.038 0 . 0 3 2 0.032 0 . 0 2 9 nei g h b o r ) Intraplanar 0.05 0.039 0.038 0.0 3 1 0.032 0.0 2 7 (1-4 n e i g hbor) Planar 0 0.02 0.011 0.010 0 . 0 0 9 0.009 0.0 0 8 groups(A) Chiral 0.15 0.129 0.1 2 4 0 . 1 0 6 0.112 0.0 9 9 centers(A3 ) Torsion angles(deg) Staggered 15 18.1 17.9 17.2 17.5 17.1 (e.g.aliphat ic Xi) Transverse 45 30.4 30.6 29.1 30.0 30.4 (e.g.aromat ic X,) Contacts(A) S i ngle 0.3 0.18 0.18 0 . 1 8 0.17 0.17 t o r s i o n Multiple 0.3 0.26 0.26 0.26 0.26 0.25 t o r s i o n Thermal factors(A?) Mainchain 1.75^ 2.12 2.05 1.81 1.75 1.48 b o n d (1-2 n e i g hbor) Mainchain 2.25 3.36 3.25 2.82 2.78 2.48 a n g l e ( l - 3 neighbor) Side chain 2.00^ 2.63 2.90 2 . 5 4 2.71 1.79 b o n d Side chain 2.503 4 . 0 4 4.38 3.79 4.00 2.93 angle 0 Max. r e s o l u t i o n (A) 2.5 2.8 2.5 2.5 2.6 No. of reflections 18160 12652 18193 18098 16312 No. of cycles 16 20 23 17 28 Final R-factor^ 0.219 0.205 0 . 2 3 0 0.223 0. 2 3 2 r - 0.02 for TP51 and HG48. 2a - 1.50 " 3 Figure 4.7 The refined structures of wild —type and mutant TyrTS's a. Shown in black are the mainchain and side chain atoms of the wild —type TyrTS, residues 46 — 54, with the exception of residue 51 shown in red for comparison with the position 51 mutant structures. The coordinates of the adenosine moiety of the bound tyrosyl adenylate are shown in blue for clarity. b. The refined coordinates of the TP51 mutant. Residues 46 — 54 and the tyrosyl adenylate shown as previously described. MOM 601 is shown as two concentric black circles. The coordinates for the proline residue at position 51 are shown in red. The R — factor is 21.1% at 2.5X resolution. 113 Figure 4.7 (continued) c. The refined coordinates of the TA51 mutant. The coordinates for the alanine residue at o position 51 are shown in red. The R — factor is 20.5% at 2.8A resolution. d. The refined coordinates of the TS51 mutant. The coordinates for the serine residue at position 51 are shown in red and HOH 601 is shown as two concentric black circles. The R — factor is 22.3% at 2.5A resolution. 114 Figure 4.7 (continued) e. The refined coordinates of the TG51 mutant. The coordinates for the glycine residue at position 51 are shown in red and IlOII 601 is shown as two concentric black circles. The R — factor is 23.0% at 2.5X resolution. / . A comparison between the mainchain and side chain coordinates of residues 45 — 55 in the wild —type TyrTS (shown in black) and the TG51 mutant (shown in red) structures. 115 Figure 4.7 (continued) g. The refined coordinates of the HG48 mutant. The coordinates for the glycine residue at position 48 are shown in red. A new water molecule, HOH 604 shown by two concentric black circles, is located near the space originally occupied by the imidazole ring of IIis48 in the wild —type molecule. The R — factor is 23.2% at 2.6A- resolution. h. A comparison between the mainchain and side chain coordinates of residues 45 — 55 of the wild-type TyrTS (shown in black) and the HG48 mutant (shown in red) structures. 116 Table 4.4 R.M.S. Differences in X between the atomic positions of residues 45—55 (mainchain and side chain) of wild —type TyrTS and each mutant TP51 TA51 TG51 TS51 HG48 Residue NO main side main side main side main side main side HIS 45 0.07 0.14 0.14 0.22 0.20 0.29 0.10 0.09 0.09 0.14 ILE 46 0.06 0.20 0.18 0.21 0.28 0.47 0.12 0.24 0.15 0.24 GLY 47 0.14 0.12 0.28 0.17 0.20 HIS 48 0.11 0.11 0.14 0.18 0.32 0.30 0.16 0.17 0.29 LEU 49 0.09 0.15 0.06 0.24 0.36 0.34 0.18 0.18 0.25 0.25 ALA 50 0.14 0.11 0.16 0.09 0.50 1.07 0.09 0.33 0.27 0.71 THR 51 0.14 0.30 0.66 0.21 0.32 0.49 ILE 52 0.11 0.10 0.08 0.26 0.31 0.32 0.18 0.26 0.26 0.35 LEU 53 0.06 0.08 0.10 0.19 0.22 0.27 0.10 0.10 0.22 0.32 THR 54 0.06 0.11 0.15 0.18 0.15 0.23 0.09 0.12 0.12 0.25 MET 55 0.16 0.19 0.17 0.18 0.11 0.37 0.16 0.22 0.07 0.24 117 Table 4.5 Temperature factors of mainchain atoms of residues 45 to 55 in wild —type and mutant TyrTS's Atom Res. No Native TP51 TA51 TG51 TS51 HG48 N HIS 45 9.3 11.0 9.3 13.3 9.6 13.4 CA HIS 45 9.3 10.6 9.3 13.3 9.3 13.5 C HIS 45 6.8 10.2 9.1 13.6 8.3 13.7 0 HIS 45 8.1 10.3 10.9 14.8 8.8 14.4 N ILE 46 9.0 11.4 11.7 14.2 9.7 15.2 CA ILE 46 10.3 12.7 13.9 14.3 10.3 16.6 C ILE 46 9.6 12.6 15.0 14.0 10.5 16.7 0 ILE 46 11.2 11.1 14.6 17.1 10.7 18.0 N GLY 47 7.0 7.4 13.0 9.4 6.9 14.8 CA GLY 47 5.2 3.1 10.7 10.1 3.8 14.8 C GLY 47 8.1 5.4 11.3 12.0 5.6 16.1 0 GLY 47 11.3 7.8 12.1 16.3 8.2 17.7 N HIS* 48 5.6 8.5 7.6 10.5 6.3 16.0 CA HIS* 48 9.4 10.6 5.9 12.3 6.8 17.7 C HIS* 48 6.5 11.5 7.9 12.4 6.9 20.3 0 HIS* 48 10.4 16.0 10.0 16.1 10.4 21.4 N LEU 49 7.4 10.7 6.4 11.5 5.7 19.1 CA LEU 49 7.7 8.8 3.6 9.8 5.3 17.2 C LEU 49 5.9 7.8 2.2 10.7 5.1 16.7 0 LEU 49 6.2 7.8 3.9 10.3 5.0 16.6 N ALA 50 5.9 7.1 2.8 10.1 4.0 16.2 CA ALA 50 8.9 8.2 4.3 11.4 5.9 16.1 C ALA 50 6.7 7.2 5.5 11.5 7.0 16.7 0 ALA 50 8.5 7.7 3.7 13.8 7.5 17.0 N THR* 51 2.7 5.1 5.2 10.5 5.4 15.4 CA THR* 51 9.0 8.6 7.3 13.2 22.0 17.1 C THR* 51 9.5 9.5 10.8 13.4 10.8 17.6 0 THR* 51 17.5 14.2 13.0 19.8 9.8 20.2 N ILE 52 10.9 12.4 13.6 18.2 11.3 18.6 CA ILE 52 11.4 13.5 12.9 15.9 10.8 18.2 C ILE 52 10.4 13.6 13.0 16.1 10.4 16.7 0 ILE 52 8.5 13.2 16.6 16.6 10.3 15.4 N LEU 53 8.5 11.5 11.7 14.1 7.9 15.8 CA LEU 53 6.5 7.6 8.9 13.2 6.4 14.2 C LEU 53 9.0 7.7 9.4 13.9 7.4 13.7 0 LEU 53 7.5 6.0 11.4 15.9 6.0 12.5 N THR 54 8.3 8.1 7.1 13.4 8.0 13.7 CA THR 54 7.2 7.7 6.6 11.6 8.2 13.2 C THR 54 8.0 8.9 10.9 11.8 8.5 13.4 0 THR 54 7.4 12.2 11.9 15.3 10.2 14.6 N MET 55 8.3 9.6 13.3 11.2 9.2 13.2 CA MET 55 12.4 14.1 15.2 14.5 13.4 15.6 C MET 55 12.3 15.4 15.6 17.3 13.8 17.1 0 MET 55 14.8 16.5 14.9 19.7 14.5 19.2 * Atoms of residue in Native 118 truncated mutant structure of the TyrTS which appeared to be displaced in the TP51 mutant structure. Evidence of movement of this water molecule, named WAT—330, disappeared upon later refinement of the TP51 structure. The remaining six water molecules in the TP51 structure are either located at positions distant from residue 51 within the ligand—free space of the active site or on the exposed surface of the molecule. The coordinates and temperature factors of the new water molecules and the new atoms in the proline residue are given in Appendix n. In general, the TA51 core structure displayed small, relatively insignificant structural alterations compared to the wild—type structure. The largest shift in coordinate position between residues 45 and 55 occurred around the mainchain atom positions of residue 51 which shifted by 0.30A (Table 4.4). This appears to be caused by a 0.6A movement of the C0 group of the alanine, relative to the C0 group of the original threonine residue. The shift is in the direction of the wild—type Thr51 O7I group (see Figures 4.6a and 4.6c for comparison) and probably occurs because this new alanine residue is no longer constrained to form a hydrogen bond with the carbonyl oxygen of residue 48. Examination of the difference distance matrix in Appendix m also shows some mainchain movements in short sequences of the structure well removed from the site of the mutation. In general, these sequences occur in exposed regions of the molecule which possess higher temperature factors (25—40A^) compared to those seen in the core of the structure (2—25A^). It is very difficult to assess the origins and significance of these structural shifts which may actually be related to the poorer quality of crystals which happened to be obtained for this particular mutant (maximum resolution = 2.8X compared to 2.5A for other position 51 mutants, Table 4.3). In addition, consistent with previous crystallographic data by Monteilhet and Blow (1978) and more recently Brick and Blow (1987) the tyrosine substrate (present at high concentrations during crystallization) appeared fully occupied in both subunits of the dimer. The atomic temperature factors are similar to those of adjacent side chains in a range characteristic for the core of the structure (listed in Appendix II). The TS51 mutant structure, Figure 4.7d, also showed no significant structural 119 alterations associated with mainchain and common side chain atoms when compared to the wild—type TyrTS, despite the addition of water molecule HOH 601 and a fully occupied tyrosine substrate. In fact, the TS51 mutant structure displayed the lowest shifts in temperature factors of any mutant structure in this study shown in Table Am .l which is located at the end of Appendix in. The mean values of temperature factors were only 0.75 A^ greater than the values for wild—type for mainchain atoms and 0.19X2 lower for side chain atoms. Alterations of common atomic positions and the pattern of temperature factors in the 45/55 segment also showed no unexpected deviations. However, the position of the C/3 and O7 groups of the new serine residue relative to the C/3 and O7I groups of the wild—type threonine residue have shifted by .70X and 1.6X, respectively. These changes are related, in part, to a rotation in Xi from 65° for Thr51 to —61° for Ser51. In the ligand—free wild—type structure, the O7I group of Thr51 is within hydrogen bonding distance (2.74X) of the carbonyl oxygen of His48 located in the same helical structure (Figure 4.7a). Such intrahelical hydrogen bonding to oxygen atoms in the preceding turn of a helix occurs with at least 85% of threonine residues in helices (Gray and Matthews, 1984) hence the conformation of Thr51 is not atypical. Serine residues in helices, however, are not so restricted. A recent analysis of the relationship between side chain conformation and secondary structure has shown that nearly one half of serines in helices of the structures sampled displayed x-\ angles near —60° (McGregor et al., 1987). Rotation of the Ser51 O7 away from the ligand—free space of the active site places it 4.3X from the carbonyl oxygen of His48. Its nearest neighbor is a weakly associated water molecule, HOH 327, located 3.8A away in the ligand—free space of the active site. The Fmut — Fwild—type electron density difference map of the TG51 mutant had indicated a series of mainchain movements from position 51 through position 49 (see section 4.5.3, Figure 4.6b). Refinement of this mutant structure showed that the movements were localized around this region of the 45/55 segment with the greatest shift appearing in the alanine side chain of position 50 which moved 1.07X (Table 4.4). Figure 4.7f shows a comparison of the refined coordinates of the 45/55 segment in wild—type (shown in black) and the TG51 mutant (shown in red). Although the 120 entire segment is sensitive to the mutation at position 51, the main movement which originates at position 51 appears to propagate its effects towards the amino terminal end (residue 45) of this structure. The mainchain shifts are also accompanied by a general increase in disorder as can be seen by the elevated values for the temperature factors in this region of the structure (Table 4.5). Because glycine lacks a 0 —carbon it has more backbone conformational flexiblity than any other residue. Substitution of a residue by glycine as in the TG51 mutant would also imply that a greater configurational entropy could be associated with the region in which the change occurs. This idea (discussed recently by Matthews et al., 1987) is consistent with the observed structure of the 45/55 segment. Examination of the difference distance matrix also shows mainchain movements associated with the carboxyl terminal portion of the molecule, residues 220—319, distant from the mutational site. In the wild—type structure this group of residues is less ordered than the core and subsequently more sensitive to positional errors during the refinement process. Alterations in this region of the TG51 structure are probably related to a general increase in disorder associated with the quality of the crystal and diffraction data as suggested by the large overall differences in temperature factors (2.96A?; Table Am .l). The TG51 structure also includes the addition of three water molcules, HOH 601 and two others located in the ligand—free space in the active site, and a fully occupied tyrosine substrate (Appendix n). The HG48 mutant, shown in Figure 4.7g, also diplayed movements of the mainchain structure associated with the complete removalof the histidine at position 48. These movements are assumed to be related to an increased configurational entropy associated with the presence of the glycine residue as previously described for the TG51 mutant. The greatest shift is again at the side chain of Ala50 which moves 0.71 A although the position of the threonine side chain at position 51 moves by nearly 0.5X (see Table 4.4). This can be seen in the comparison of the 45/55 segment between the wild—type and HG48 mutant structures shown in Figure 4.6h. As was also seen in the TG51 mutant structure the region of the structure most sensitive to the mutational change is located between residues 48 and 51. The patterning of the 121 temperature factors also indicates a general increase in disorder throughout the entire 45/55 segment. The biological significance of these effects as related to the activity of these two mutants (TG51 and HG48) are discussed throughout Chapter 5. In addition, removal of the imidazole ring in this mutant also allows a water molecule, HOH 604, to occupy this open space (shown in Figure 4.7g). HOH 604 is within hydrogen—bonding distance of the amino group of residue 45 and has a reasonably low temperature factor of 24.9 A^ (Appendix II). This particular water molecule may not necessarily be displaced in the presence of enzyme—bound tyrosyl adenylate and may play a role in decreased stability of the tyrosyl adenylate to hydrolysis (discussed in section 5.5.2). Tyrosine was also observed at full occupancy in the HG48 mutant structure. A discussion of the conflict between this observation and the kinetic studies which indicate that only half of the active sites are occupied in solution follows. 4.7 Discussion The structural results can be summarized as follows: 1. The refined crystal structures of the TP51, TA51 and TS51 showed no significant mainchain or common side chain structural differences when compared to the wild—type structure. 2. The refined structure of the TP51 mutant shows that the postulated movement of WAT—330 described by Brown et al. (1987) does not exist. 3. Large coordinate shifts, from 0.6A to 1.5A, were observed for the C/3 group of Ala51 in the TA51 structure and the C|3 and O7 groups of Ser51 in the TS51 structure when compared to the Thr51 residue in the wild—type structure. These shifts appear to be related to the chemical nature of each residue in its local enviroment and its movements do not appear to significantly propagate throughout the rest of the structure. 4. Complete removal of the threonine side chain in the TG51 crystal structure and the histidine side chain in the HG48 crystal structure appears to have caused a series of mainchain and side chain movements affecting, in particular, residues 48 to 51. The greatest shift appeared in the side chain of Ala50 which moved 1.07A or 0.70A, 122 respectively. 5. The only significant solvent rearrangement occurred in the HG48 crystal structure where a new water molecule (HOH 604) with a reasonably low temperature factor has appeared near the space previously occupied by the imidazole ring of the histidine. 6. In all structures where tyrosine was present at high concentrations during crystallization, electron density for the tyrosine substrate appears in both subunits of the dimer at a stoicheometry approaching 2 mol of tyrosine/mol of dimer. The biological significance of the results in items 1—3 as they relate to the catalytic function of the TyrTS are discussed throughout the next chapter. The apparent conflict with the result in item 4 with measured kinetic parameters will be discussed here Crystal structures of the unliganded wild—type and the enzyme—tyrosine complex of the wild—type TyrTS have shown that the dimer is composed of two symmetrical subunits (Monteilhet and Blow 1978; Brick and Blow, 1985). Furthermore, in the crystal structures of the wild—type and truncated TyrTS the tyrosine substrate appears at occupancies similar to those observed for the mutant structures presented here. In contrast, the results of equilibrium dialysis experments have found that only one mole of tyrosine binds to each mole of enzyme dimer (Bosshard et al., 1975; Fersht et al., 1975). More recently, kinetic studies by Ward and Fersht (1988) have provided additional evidence for this apparent asymmetry of tyrosine binding and have provided new evidence which implies that the ligand—free wild—type enzyme is inherently asymmetrical in solution. This apparent conflict of results does not necessarily indicate a fault with either set of experimental data but rather arises from the fact that crystallographic studies to date have not been able to substantiate the half—of—the—sites activity observed in solution. It is therefore worth questioning how both experimental results can exist without invalidating each other. Monteilhet and Blow (1978) have discussed various reasons for the origins of their structural data which indicate an occupancy of tyrosine at 76% in each monomer unit. The first possibility is that the dimer might lose its symmetry and remain undetected by crystallographic studies. In the wild—type molecule the crystal symmetry imposes a 2 —fold symmetry on the image of the dimer which, in this structural determination as 123 well as those presented in this thesis, preserves the crystal symmetry in a statistical sense. However, in the refined structure of the truncated TyrTS mutant each monomer provided an independently refined model of the tyrosine binding site (Brick and Blow, 1987). In this case the binding of tyrosine in each site appeared identical to that of the wild—type structure. It is therefore unlikely that a detectable difference in structure of each site in the full length wild—type and mutant structures is remaining undetected. Another possibility is that crystallization selects and 'locks' a symmetrical conformation of the TyrTS (Monteilhet and Blow, 1978). This theory could explain the differences in stoicheometries of tyrosine binding if an asymmetrical form of the enzyme truly exists as implied by the work of Ward and Fersht (1988). Those studies showed that each dimer was active at only one site of each monomer and that essentially no interconversion occurred between an active and inactive site over several minutes when the enyme is turning over in the steady state. However, once TyrTS is crystallized with tyrosine and then redissolved in solution active site titration of this material gives a figure of 1.6 active sites per molecule consistent with the crystallographic data (Monteilhet and Blow, 1978). This result can be consistent with the kinetic studies if the time scale of the conversion between the asymmetrical and symmetrical forms is very slow. It is also possible that the high tyrosine concentration (ImM) during crystallization may induce the symmetric form of the dimer although Monteilhet and Blow (1978) had shown that the incorporation of tyrosine into crystalline TyrTS approached a stoicheometry of two moles tyrosine per mole enzyme. A third possibility is that a second weak binding site for tyrosine has escaped detection under the experimental conditions used for equilibrium dialysis (Bosshard et al., 1975). The feasibility of this theory is shown in the Scatchard plot in Figure 4.8 which displays the theoretical curve for two binding sites with very different dissociation constants. The associated errors with this data are also shown for two typical enzyme concentrations used in this type of experiment. The best straight line for points on this curve representing the typical range of tyrosine concentrations (5—200jjM; Wells, 1987) used in this experiment gives a value of 1.1 (1 within error) 124 Figure 4.8 Scatchard plot of two non—identical binding sites A theoretical Scatchard plot of the binding of a ligand (i.e. tyrosine) to two non—identical sites based upon the following equation: r - c/(Kt , + c) + c/(Kj 2 + c) where x - r and y - r/c r is the number of moles of ligand (A) bound to one mole of macromolecule (P) and defined as: r = [AJbound/fPltotai, c is the concentration of the ligand in /*M, and A y, = 2 0 and A j 2 = 1000/xM are the dissociation constants for the strong and weak binding sites, respectively. In this diagram theoretical points on this graph are shown as open circles. The solid line represents the best straight line for points obtained using standard experimental conditions. In an equilibrium dialysis experiment the difference of concentration of ligand between the two chambers at equilibrium is: r[P]totai = (c + r[P]total) “ c- Assuming a measurement error of 2% in c and (c -♦* r[P] total)* the percentage error in r for points which deviate from the straight line in the Scatchard plot is shown below: c r %error in r terror in r 60 0.807 17 4.9 100 0 . 9 2 4 24 6.3 200 1.076 39 9.4 500 1.295 81 18 1000 1.480 137 29 125 tyrosines bound per active site (dimer). However, as the curve deviates from the linear fit at tyrosine concentrations greater than 200/(M, the errors associated with these points for both enzyme concentrations is so large that they would statistically be discarded and hence evidence of a second weak binding site could easily go undetected. This possibility is implied in the paper by Bosshard et al. (1975) which shows equilibrium dialysis data taken to ImM tyrosine concentrations. Fersht (1975) has also shown that in the presence of ATP and inorganic phophatase (to allow the accumulation of tyrosyl adenylate) that two moles of tyrosine bind to the enzyme to form the [E*Tyr—AMP, ATP, Tyr] complex (K§ = 144/iM for the weaker binding site). In contrast, the binding curve representing the incorporation of two moles of tyrosine into one mole of crystalline TyrTS showed no sign of any interaction between the two sites (Monteilhet and Blow, 1978). This result, though, is not necessarily inconsistent with the kinetic results if one accepts the existence of both symmetrical and asymmetrical forms of the enzyme. In summary one has to accept the results of both sets of kinetic and crystallographic studies as neither have been designed with the purpose of proving the other wrong. More importantly, neither crystallographic structures nor kinetic results have provided any information from which to speculate about origin or magnitude of structural differences which could be responsible for the apparent asymmetrical activity of the TyrTS enzyme. A possible resolution to this 'structure versus function paradox' must await the interest of future researchers. 126 CHAPTER 5 DISCUSSION 5.1 The Thr51-»Pro Mutation 5.1.1 Introduction The initial aim of this study was to understand the relationship between observed changes in catalytic function and observed changes in three-dimensional structure in the enzyme tyrosyl—tRNA synthetase (TyrTS) from Bacillus stearothermophilus. The residues of the active site of the enzyme which interacted through potential hydrogen bonds with the intermediate tyrosyl adenylate were identified from the corresponding crystal structure (section 4.1.1; Blow and Brick, 1985). A quantitative description of the contribution of each hydrogen bonding group to specificity, binding and/or catalysis was determined through a series of protein engineering experiments (see Fersht, 1987 for a general review). These experiments systematically deleted, using site—directed mutagenesis techniques, each identified hydrogen bonding donor or acceptor in the active site. The rate and equilibrium constants for each step in the overall reaction scheme were measured for each mutated enzyme and used to determine the corresponding energetic contributions in terms of Gibbs' free energies of each side chain (Fersht, 1974, 1985). In particular, the hydroxyl group of the threonine residue at position 51 appeared to make a polar interaction, possibly through a long hydrogen bond, with the ribosyl ring oxygen ( 0 —1) in the original wild—type crystal structure (Monteilhet and Blow, 1978; Rubin and Blow, 1981; Bhat et al., 1982). Later studies refined this distance to 3.6A(±0.2A) (P. Brick, personal communication) asserting that no hydrogen bond was formed (Brown et al., 1987). Consistent with the methodology previously described for determining the energetic contribution of this particular group, this residue was mutated to an alanine (Wilkinson et al., 1984). In addition, of the nine residues originally identified as having potential hydrogen bonding interactions with the tyrosyl adenylate 127 position 51 was the only residue not conserved from the three known sequences of bacterial tyrosyl—tRNA synthetases. Notably in the E. coli enzyme (56% homologous) position 51 (Winter et al., 1983) but in the B. caldotenax enzyme (99% homologous) the residue was a or /cm )>i^ (Jones at al., 1986). Hence, to explore the consequences of the sequence variance at this position threonine 51 was also mutated to a proline (Wilkinson et al, 1984). 5.1.2 Kinetic Characterization Compiled kinetic data of mutants of the TyrTS demonstrate that deletion of a side chain on the enzyme that forms a good hydrogen bond with an uncharged group on the substrate weakens binding by 0.5—1.5kcal/mol (Fersht et al., 1985). However, in the case of the Thr51-»Ala mutation a hydroxyl group was deleted which was supposed to be involved in such an interation with the tyrosyl adenylate (see previous section). Pyrophosphate exchange kinetics (section 3.5), however revealed a stabilization of the enzyme—substrate interaction energy by 0.38kcal/mol. Furthermore, the Thr51->Pro mutation resulted in a stabilization of 1.9kcal/mol (Wilkinson et al., 1984). This followed from an observed 15—fold decrease in Kj^ATP) combined with a near doubling of fccat which lead to an overall 25—fold increase in the measured ^cat^M - The original pyrophosphate exchange data for both the TP51 and TA51 mutant are shown in Table 5.1. The free energy profiles for both mutants and the accompanying difference energy diagram for the TP51 mutant are shown in Figures 5.1a and 5.1b, respectively (Ho and Fersht, 1986). The stabilization of the activated complex for the TA51 and TP51 mutants, 0.41 and 2.17kcal/mole, respectively, was very similar to that measured for the steady—state reactions. Specifically, the apparent binding energies for these mutations stabilized the enzyme—tyrosyl adenylate complex (E-Tyr—AMP) by 0.96(TA51) and 2.30kcal/mol(TP51) in the transition state. As the reaction proceeded there continued to be an incremental rise in the binding energies of all subsequent intermediates which involve bound forms of the tyrosyl adenylate. It is essential to note that there is virtually no difference in the ability of these mutants to bind either 128 Table 5.1 Pyrophosphate exchange activity of wild—type and position 51 mutant TyrTS's Enzyme * cat ^cat/^M ( s ' 1) (mM) TyrTS(wi Id-type)^- 8.35 1.08 7730 TP512 12.0 0.058 208000 TC511 1 2 .4 0 .35 35400 TA512 8.6 0 .5 4 15900 TG511 6 .0 1.25 4800 TS511 1.88 1.16 1620 TyrTS exchange activity was measured at 25°C, pH7.8, in 144mM Tris—HC1, lOmM MgCl 2, 0.1 mM PMSF, lOmM 2—ME, 2mM pyrophosphate and 5 0 tyrosine. Table 5.2 Aminoacylation activity and the Gibbs' free energy of the enzyme—tyrosyl adenylate complex of TyrTS's Enzyme * cat *M TyrTS(wild-type)1 4 .7 2 .5 1860 -5 .9 9 TP512 1.8 0.019 95800 -8 .2 9 TC511 2.9 0.29 8920 -7 .4 3 TA512 4 .0 1.2 3200 -6 .9 5 The same conditions were used for the determination of aminoacylation activity as in Table 5.1, but with 100/xM tyrosine and 0.002 unit/ml inorganic pyrophosphatase. iFersht et al., 1985. 2Wilkinson et al., 1984. 3H o and Fersht, 1986. 129 Figure 5.1 Gibbs' free energies of enzyme —bound complexes of position 51 mutants (taken from Ho and Fersht, 1986) so 60- [B.T-A ] * 40- 2 o- oo- -2 0- - 40- - 60- -SO- - 10 0- - 12 0 - Q koal m o l'1 TyrTt(Cys81) TyrTSOUoSD TyrTtCProfl) a. The Gibbs' free energy profiles for the formation of tyrosyl adenylate (as defined by Scheme I; section 3.6.1) by wild—type and each indicated mutant. The values for these free energies and the free energy differences between mutant and wild—type enzyme—substrate complexes are given in Tables 5.4 and 5.5. 4f a n CL < < kcal mol*1 < » 1 i AG ►: £ HI- uj “ u i ui 0 .0 1.0 2.0 3 . 0 b. The Gibbs' free energy differences of enzyme —bound complexes of the TP51 mutant relative to those of the wild—type TyrTS enzyme. 130 tyrosine or ATP compared to the wild—type enzyme. For this reason they have been classified as differential binding mutants according to the conventions of Albery and Knowles (1976). Regarding the aminoacylation of tRNA (the second step of the reaction) it was pointed out by Mulvey and Fersht (1977) that the TyrTS enzyme exists mainly as the tyrosyl adenylate complex (E*Tyr—AMP) under physiological conditions. In the case of the TP51 mutant and to a much lesser extent the TA51 mutant, though catalytically efficient in forming the tyrosyl adenylate complex, in vivo these enzymes would be less efficient in forming tyrosyl —tRNA because the enzyme bound adenylate would constitute a free energy minimum or a 'thermodynamic trap' (Ho and Fersht, 1986). The aminoacylation activity data and the Gibbs' free energy of the enzyme-tyrosyl adenylate complex for mutant and wild —type TyrTS's are given in Table 5.2. 5.1.3 The Double Mutant Test Of all mutations made throughout the TyrTS enzyme only changes at position 51 yielded an improvement in substrate binding and enzymic catalysis. However, the choice of which mutant to study using crystallographic techniques was based in part upon results obtained from the 'double mutant test' (Carter et al., 1984; Lowe et al., 1985). The original aim of the double mutant test was to introduce a second mutation in the active site in order to understand how the first mutation affected substrate affinity. To do this three mutant enzymes had to be constructed: two, each containing a single mutation, and a third containing both mutations simultaneously. The kinetic parameters for each mutant enzyme had to be measured to determine the overall change in Gibb's Free energy for the activation reaction. The complete scheme, with regard to the thermodynamic relationships, is shown at the top of the following page (Carter et al., 1984). 131 If there was no interaction between the two mutations (via structural alterations or solvent rearrangements for example, which could be observed kinetically) then the measured Gibb's free energy of the enzyme—substrate transition state of the doubly mutated enzyme would simply be the sum of the corresponding terms of the two singly mutated enzymes. The following relationship also holds: AG, — AG, and AG 2 = AG 2 Such a relationship was described between the Cys35-*Gly and the His48->Gly mutations illustrated in Figure 5.2a. These residues are spatially separated in the active site by approximately 10.4A. They appear to interact with different portions of the tyrosyl adenylate and it was postulated that any changes in the binding of the substrates/intermediates were not communicated between the two sites. However, if there was an interaction between the two mutated sites then the measured interaction energy for the doubly mutated enzyme would not equal the sum of the corresponding terms for the two singly mutated enzymes. In other words: AG, ?£ AGj and AG 2 ^ AG2 AG, + AC2 - AG\ + AG2. In this case, because (AG, — AG,) = AG2 ~ AG2 ^ 0 then the term |AG, — AGji, defined as the coupling energy between the two mutations, would be a measure of the energetic difference between the double mutant and each single mutant. When the structural changes do not interact, as in the Cys35-*Gly and His48-*Gly mutations described previously, the coupling energy would be 132 Figure 5.2 The energy of interaction of each side chain with the transition state(kcal/mol) in the amino acid activation reaction (taken from Carter et al., 1984) + 1-2 kcal Cys35 His48 ------Gly3S His48 s ^ oos ^ 'm ’1 1.120s’ 1M’1 + m + 1-1 kcal kcai Cys35 Gly48 Gly35 Gly48 + 1*2 kcal 1 7 7s *’ m " 1 a. The non—interactive relationship between the Cys35-»Gly and the His48-*Gly mutations. His48 Thr51 -■ 9 kc» His48 Pro 51 s ^ o o s -’ m *1 208,000 s"1M‘1 + 1*1 kcal + 3 0 kcal G ly48 Thr51 ------» 0-0 kcal J G/y 48 Pro 51 1 4 6 0 s ’1m ’1 1,380 s^M*1 b. The interactive relationship between the His48-»Gly and the Thr51-»Pro mutations. 133 zero. However the same test was applied to the Thr51->Pro mutation in conjunction with the His48-*Gly mutation (see Figure 5.2b) and a coupling energy of 1.9kcal/mol was observed. These two residues are located on the same portion of a —helix maintaining a spatial separation of approximately 5.7A. The original interpretation was that the proline at position 51 was distorting the a —helix in some manner so as to affect the interaction of the histidine at position 48 with the tyrosyl adenylate (Carter et al., 1984). Additional studies on double mutants which replaced His48 with asparagine and glutamine (Lowe et al., 1985) emphasized that the presence of a hydrogen—bond donor at position 48 appeared to be essential for the proposed structural change induced by the TP51 mutation to bring about an improvement in the transition—state stabilization. A crystal structure of the TP51 mutant was undertaken to confirm this theory. 5.1.4 The Crystal Structure In the preliminary unrefined structure of B. stearothermophilus tyrosyl—tRNA synthetase (Bhat et al., 1982), residues 46 —60 were assigned as o —helical. A more detailed study, confirmed later by refinement (Brick and Blow, 1987; Brick, Bhat and Blow, in preparation), showed the amino terminus of this helix to be irregular although the conformational angles all lie near to or within the a —helical region of a Ramachandran plot (Figure 5.3; Ramachadran et al., 1963). In particular, NH51 appears to make no hydrogen bond to the mainchain and the CO46...NH50 interaction is through a well-ordered water molecule (shown in Figure 5.4). Consistent with this observation, the refined structure of the TP51 mutant (section 4.6.3) showed no structural distortion of the mainchain or for that matter any side chains which could have conceivably been affected by the change (Figure 5.4). This is further illustrated by the electron difference density map of the TP51 mutant, Figure 4.6a, which shows only difference density near the mutation site. Section AIQ.l of Appendix m also shows plots of the relative movements in A of mainchain and side chains between the refined TP51 and wild—type enzyme crystal structures. The postulated movement of His48 was not observed in this ligand—free enzyme. 134 Figure 5.3 The Ramachandran plot of residues 45-60 of the wild-type TyrTS structure In this diagram solid lines enclose 'normal' contour regions for a —helical and 0 —pleated sheet structures. Dotted lines enclose 'outer —limit' contour regions. All residues are denoted by a X except glycine which is shown by a □ The corresponding values are listed below. residue Phi Psi His45 -140 -179 Ile46 -63 -14 Gly47 -71 -24 His48 -95 - 8 Leu49 -63 -38 Ala50 -63 -52 Thr51 -63 -42 Ile52 -5 6 -54 Leu53 -6 9 -31 Thr54 -6 4 -38 Met55 -5 8 -45 Arg56 -5 7 -44 Arg57 -6 5 -40 Phe58 -67 -46 Gln59 -6 0 -3 9 Gln60 -63 -29 135 Figure 5.4 Hydrogen bonding in the wild-type and TP51 mutant TyrTS structures eur«r 0LY47 icu« LEU32 Mainchain atoms of wild—type TyrTS, residues 46—54, with the adenosine moiety shown for clarity. The side chain for Thr51 is shown and the side chain of Pro51 is indicated in thinner bonds. The dashed lines indicate close polar interactions which are stereochemically favorable for hydrogen bonds in the refined structure. Beginning with the C048...NH52 interaction, regular a —helical hydrogen bonds are made, but NH51 makes no hydrogen bond to the mainchain, and the CO46...NH50 interaction is through a well-ordered water molecule, indicated as two concentric circles (taken from Brown et al., 1987). 136 5.1.5 Discussion It becomes apparent upon recalling the experimental observations made so far that many or possibly most of the original conjectures about the origins of the effects caused by this mutation need reexamination. The 'uninterpreted' experimental results are summarized as follows: 1) The TP51 mutant enzyme displays a 25—fold increase in activity for the activation of tyrosine compared to the wild—type enzyme. There is essentially no difference observed in this mutated enzyme's ability to bind tyrosine or ATP compared to the wild—type enzyme. However, all complexes involving enzyme—bound tyrosyl adenylate (the transition state and all subsequent intermediates) demonstrate improved energetic stabilization compared to the wild—type enzyme. 2) Results from the double mutant test indicate that there is some type of interaction between the mutated and/or wild—type residues present at positions 48 and 51. 3) The refined crystal structure of the enzyme—bound tyrosyl adenylate shows the distance from the hydroxyl of the threonine at position 51 to the 0 —1 ring oxygen of the tyrosyl is 3.6A(±0.2A). ■4r) The crystal structure of the ligand—free TP51 mutant provides no indication of a mainchain or side chain distortion introduced by the presence of a proline in this slightly distorted a —helix. The experimental problem, however, remained the same. That is, there existed no reasonable explanation to account for the nature of the high catalytic efficiency of the TP51 mutant; especially when compared to mutants whose activities fell between TP51 and the wild—type enzyme. Most of the remaining portion of this chapter describes the development and subsequent testing of theories which attempt to rationalize the earlier structural and functional experimental observations made regarding this mutant. The significance of this work in the current Held of protein engineering is discussed in the final summary section (5.6) of this chapter. 137 Table 5.3 Contributions of individual groups at position 51 to binding energy of ATP in the transition state (taken from Fersht et al., 1985) contribution to binding energy group (kcal/mol) 7 -methyl -0.92 7 -SH -0.46 7 -OH +1.36 /3-methyl -0.72 138 5.2 The Position 51 Series 5.2.1 Kinetic Characterization Based upon the first experimental results of enhanced catalyic activity displayed by the TP51 and TA51 mutants (section 5.1.2; Wilkinson et al., 1984) a series of mutations were constructed at position 51 to further investigate possible effects on substrate binding and catalysis (Fersht et al., 1985). This particular study had, in effect, two aims. The First was to the determine energetic contributions of the various component atoms of the wild—type threonine side chain. The second was to determine the significance of the spatial separation between the hydroxyl group of threonine—51 and the ribosyl ring oxygen of the tyrosyl adenylate substrate. Initially, the wild—type threonine side chain was 'dissected' into its component parts through the construction of mutants with smaller amino acids at position 51, namely serine and glycine. From the steady state parameters measured using pyrophosphate exchange kinetics (Table 5.1) the changes in binding energy of ATP in the transition state were calculated. Using a series of systematic comparisons which included the steady state data from the TA51 and TC51 mutants, the contributions of individual groups to binding energy were adduced, summarized in Table 5.3. These data consistently showed that only the hydroxyl portion of the threonine—51 residue energetically destabilized the transition state. As was previously discussed in section 5.1.1 the refined distance between the hydroxyl group of the threonine—51 and the ring oxygen of the tyrosyl adenylate has been shown to be approximately 3.6A. This was interpreted by Wilkinson et al. (1984) to represent a weak unfavorable interaction. Fersht et al. (1985) predicted that replacement of the threonine with a cysteine might form a good hydrogen bond in comparison thereby implying that a polar interaction between residue 51 and the ribosyl ring oxygen might enhance the affinity for ATP. Consistent with this theory the TC51 mutation demonstrated a 50% increase in k^ for the activation of tyrosine combined with a 3—fold lowering of for ATP (see Table 5.1). In a later study (Ho and Fersht, 1986) the pre—steady state characterization of 139 Table 5.4 Gibbs' free energies of complexes of wild—type and mutant TyrTS enzymes kcal/m ol enzyme GETyr-A TP c[ETyrATP]* GETyr-AMPPPi CE Tyr-AMP wild-type^ -9.89 5.41 -1 0 .3 8 -5 .9 9 TV51 -11 .7 6 2.69 -1 3 .4 0 -8 .5 3 TP512 —10.29 3 .24 -1 2 .6 5 -8 .2 9 TC512 -1 1 .0 8 4 .17 -1 1 .5 9 -7 .4 3 TA512 -9 .8 9 5.00 -1 1 .0 5 -6 .9 5 TG513 -10.37 5.26 -10 .5 2 -5 .7 5 HC481 -9 .9 6 7.0 4 -8 .7 6 -4 .1 4 Table 5.5 Difference in Gibbs' free energy between bound complexes of each mutant TyrTS and the wild—type enzyme kcal/m ol enzyme ^ GET y rA T P ^[E-Tyr-ATP]* ^ GE Tyr-AMP PPi ^ CE-Tyr-AMP TV51 -1 .9 -2 .7 -3 .0 -2 .5 TP512 — 0 .4 -2 .1 7 -2 .2 7 -2 .3 0 TC512 -1 .1 9 -1 .2 4 -1 .2 1 -1 .4 4 TA512 0 -0 .4 1 -0 .6 7 -0 .9 6 TG513 -0 .4 8 -0 .1 5 -0 .1 4 0 .2 4 HG481 0 .4 1.2 1.2 1 .5 ^Wells and Fersht, 1986. 2Ho and Fersht, 1986. 3Wells, 1987. 140 those mutant enzymes which improved transition state stabilization revealed how each affected binding and catalysis at each step along the reaction pathway (Scheme 1; section 3.6.1). Specifically, the TAS1 and TP51 mutants were classified as differential binding mutations. This is to say, as shown in Figure 5.1 and Tables 5.4 and 5.5, both mutants distinguish between the reacted and unreacted forms of ATP by primarily stabilizing the [E-Tyr-ATP]^, E-Tyr—AMP-PPi, and E-Tyr—AMP complexes. In constrast, the TC51 mutation lowers the free energy of all states in which the enzyme is bound to the ATP moiety and was hence described as a uniform binding mutation. The mechanistic interpretation of this observation implied that the sulfhydryl group of the cysteine was making an equally favorable interaction with reacted and unreacted ATP possibly indicating that an optimal contact was maintained with the ribose moiety throughout the reaction. Although the possible improvement in ATP moiety binding can still be explained by an improved hydrogen bonding distance of the cysteine — SH group there is no kinetic information to explain why the TC51 mutant affects the binding of free ATP while the TA51 and TP51 mutants essentially do not in comparison to the wild—type enzyme. 5.2.2 Linear Free Energy Relationships (LFER's) Additional information regarding the use binding energy by mutant enzymes at position 51 throughout the course of the reaction to form tyrosyl adenylate has been obtained from the application of linear free energy relationships (LFER's). Specifically, utilizing a Bronsted or a Hammett plot one can link changes in a rate constant for a particular reaction to changes in an associated equilibrium constant (Jencks, 1985). The regression of such data to a line strongly suggests that the same reaction mechanism is being employed. In addition, the slope (0) of such a line gives an indication of the structure of the transition state. If 0 is close to 0 then the transition state resembles the starting materials whereas if 0 is close to 1 then the transition state resembles the products. Fersht et al. (1986) have shown that the activities of mutant tyrosyl—tRNA synthetases fit structure—activity relationships which relate the rate constant for the 141 Figure 5.5 Linear free energy relationships for TyrTS reactions (taken from Wells, 1987) a. b. c. log (k 3Kpp/k .3/fa') l°9 jK p p 'k *3 *V ) The mutant enzymes are signified by a one letter code in these figures. For example, the Thr51-»AIa mutation is A51 instead of the conventional TA51 used throughout this thesis. The values shown for the TV51 (V51) mutation in all figures and the value shown for the TC51 mutation in Figure 5.5a are not included in the linear regressions. The TV51 mutant data are discussed in section 5.3. 141a Figure 5.6 Changes in binding—energy for the formation of tyrosyl adenylate Reaction — » Summary of the average binding—energy changes, based upon the 0 values in Figures 5.5a—c, on going from the enzyme—tyrosine complex (ET), to the ETyr-ATP complex (ETA), to the transition state (E[TA]*) to the first intermediate, E*Tyr—AMP-PPi (ET — A.PP), to the final intermediate, E-Tyr — AMP (ET —A) (based upon a figure from Fersht et al., 1986 using data from Wells, 1987). 142 formation of enzyme—bound tyrosyl adenylate (E-Tyr—AMP) to its equilibrium constant with enzyme—bound tyrosine and ATP (E-Tyr-ATP). Fersht et al. (1987) and Wells (1987) took the analysis one step further by making the same comparison using the equilibrium constant of enzyme—bound tyrosine only in order to examine any appreciable changes in binding energy when free ATP bound to the enzyme. Fersht et al. (1986) postulated that in enzymic reactions changes in structure well removed from the seat of reaction alter binding energy. Thus the values of 0, in general, measure the fraction of total changes in binding energy (Fersht, 1985). The LFER given in Figure 5.5a, which compares the yields 0=0.10 which indicates that 10% of the binding energy change occurs upon the formation of the E-Tyr-ATP complex. The LFER's in Figures 5.5b and c show, respectively, that 81% of the binding energy change occurs on formation of the transition state (E[TA]*) and 91% occurs on formation of the E-Tyr—AMP.PPi complex (Wells, 1987). These data are summarized in Figure 5.6. With regard to the position 51 series (without the TS51 mutant which lacks pre—steady state analysis) the mutants appear, in general, to regress to the same line in all three intermediate steps of the reaction. This suggests that these mutant enzymes are binding these complexes by a similar mechamism. The only departure from linearity appears to occur with the TC51 mutant during the binding of free ATP and hence it was not included in the least squares fit of the data in Figure 5.5a. As dicussed in section 5.2.1, the TC51 mutant is the only mutant which demonstrates enhanced binding of ATP in the pre—transition state compared to the wild—type enzyme. Whether TC51 binds ATP using different interactions cannot be safely inferred from the given kinetic data and shall be further discussed in the context of additional structural and kinetic results presented in this chapter. 5.2.3 Crystal Structures The refined structure of the wild—type TyrTS complexed with an inhibitor, tyrosinyl adenylate (YRSI), which has a crystallographic R —factor of 20.4% at 2.7A resolution (P. Brick, personal communication), is used as the model from which interaction distances between the enzyme and the tyrosyl adenylate are quoted. In this structure, the hydroxyl group of residue Thr51 approaches the ribosyl ring oxygen of the inhibitor but the distance is too long for a true hydrogen bond (section 5.1.1). However, this hydroxyl group is within hydrogen bonding distance (2.74X) of the carbonyl oxygen of His48 located in the same helical structure (Figure 5.4). Such intrahelical hydrogen bonding to oxygen atoms in the preceding turn of a helix occurs with at least 85% of threonine residues in helices (Gray and Matthews, 1984) hence, the conformation of residue Thr51 is not atypical. Kinetic studies discussed in the previous section have consistently shown that the presence of an O 7 group at position 51 (in threonine or serine) destabilizes the binding of tyrosyl adenylate to the TyrTS. In the case of the Thr51 residue, if it is held in this intrahelical hydrogen bonding conformation then an unfavorable lone pair—lone pair interaction can occur between its O7 I oxygen atom and the 0 —4 oxygen atom in the ribosyl ring. However, in the YRSI structure, the electron density for Thr51 is 'rounded' thereby implying that the orientation of this residue is not so well-defined that its O 7 I oxygen would always be locked into such an energetically unfavorable conformation. As observed with the TP51 mutant structure, previously described in section 5.1.4, the crystal structure of the TA51 mutant showed no significant mainchain or common side chain structural differences when compared to the ligand—free wild—type structure. The C/3 group of Ala51 in the TA51 mutant had, however, shifted 0.6A in the direction of the the wild—type Thr51 O 7 I group. This probably occurs because the new alanine residue is no longer constrained to adopt the intrahelical hydrogen bonding adopted by the previous threonine residue. In comparison to the coordinates of the wild—type enzyme in the YRSI structure, the C/3 of Ala51 has only shifted 0.07A relative to the C/3 group of the Thr51 residue. It follows that no significant differences were observed in the contact distances between this group and the adenosine moiety of the tyrosinyl adenylate. The results suggest that the origin this mutant's enhanced activity appears to be directly related to deletion of a destabilizing interaction between the O7 I group of Thr51 in the wild—type enzyme and the tyrosyl adenylate (Wilkinson et al., 1984). 144 The only signficant structural difference displayed by the TSS1 mutant structure was an adjustment in the position of its C0 and O 7 groups of SerSl relative to the C/3 and O 7 I groups of Thr51 in the ligand—free wild—type. The shift of the O 7 groups were related, in part, to a rotation of the Xi angle from 65° for Thr51 to —61° for the SerSl residue. Rotation of Ser51 does not appreciably alter the distance between its Oy group and the ribosyl ring oxygen (3.7X) which, as discussed earlier, may represent an unfavorable lone pair—lone pair interaction. In addition, the O 7 group approaches the 0 —3 atom of the tyrosinyl adenylate (Figure 4.7d) at a distance, 3.4A, indicative of a weak hydrogen bond. This interaction may also represent another destabilizing lone pair—lone pair interaction. This can further be complicated by the fact that the 0 —3 group of the tyrosinyl adenylate appears to form a strong polar interaction with a water molecule, HOH 326, located perpendicular to the direction approached by the Ser51 O7 group. The TS51 mutant has been shown by Fersht et al. (1985) to display the poorest activity of any position 51 mutant for the formation of tyrosyl adenylate (*4.8—fold lower than the wild—type enzyme). The reason given assumes that the hydroxyl group of the serine must break its hydrogen bond with water in order to form the enzyme— substrate complex. Similarly to the wild—type case, the Ser51 O7 group was assumed to make the same poor interaction with the ribosyl ring oxygen (0 —4). The crystal structure weakly suggests that the interaction with water molecule HOH 327 may be exchanged for an interaction with the 0 —3 group of the tyrosyl adenylate. HOH 327 though, appears to make a strong polar interaction with HOH 326 which is known to form a strong polar interaction with the 0 —3 group. Hence, it is probably HOH 326 which more strongly influences the binding of the 0 —3 group to the enzyme. The crystal structure of the TS51 mutant, however, more strongly suggests that the origin of the l.lkcal mol - 1 transition state destabilization demonstrated by this mutant is related to unfavorable lone pair—lone pair interactions not only between the 0 —4 group of the tyrosyl adenylate but the 0 —3 group as well. The crystal structure of the TG51 mutant showed that complete removal of the threonine side chain appeared to cause a series of mainchain and side chain movements affecting, in particular, residues 48 to 51 (see Figure 4.7e). The shifts in mainchain coordinates were also accompanied by a general increase in disorder as shown by elevated values for temperature factors in this region of the structure. Kinetic studies have shown that the activity of TG51 for the activation of tyrosine falls between wild—type and the less active TS51 mutant (Fersht et al., 1985; see Table 5.1). Pre—steady state data confirmed a 3 —fold drop in kcat for the formation of tyrosyl adenylate although the for ATP/tyrosyl adenylate was approximately halved (Wells, 1987). This is reasonable considering glycine lacks both the C/3 and C 7 groups of the threonine necessary for stabilization of the tyrosyl adenylate on the enzyme. The TG51 mutant also lacks the strongly destabilizing hydroxyl group which appears to dominate the catalytic effects of the less active TS51 mutant. In addition, the crystal structure also shows that the decreased levels of activity of the TG51 mutant may be related to an undesirable increase in the entropy of a region of the TyrTS which contains atoms that may interact with ATP and/or enzyme—bound tyrosyl adenylate (in particular, the N51 group of His48 which may form a hydrogen bond with the ribosyl ring oxygen). The structure of the TC51 mutant has been determined with enzyme—bound tyrosyl adenylate in one case and enzyme—bound tyramine (tyrosine where the carboxylic acid moiety is replaced by a methyl group) in the other (M. Fothergill, personal communication). The R —factor for both structures is currently 23% at 2.5A resolution although refinement is still in progress. In the structure containing tyrosyl adenylate a disulfide bridge has formed between the S 7 group of the Cys51 residue and the S7 group of Cys35. The tyrosyl adenylate appears bound in a similar fashion to that observed for the tyrosinyl adenylate in the YRSI structure. This result tends to support the validity of using the coordinates of the tyrosinyl adenylate in conjunction with coordinates of the mutants presented in this thesis which do not contain this intermediate for discussion of the effect of potential enzyme—substrate interactions. In the enzyme—bound tyramine structure (coordinates kindly made available by Michael Fothergill), however, residue Cys35 is reduced and tyramine is bound in the cleft of the enzyme normally occupied by tyrosine. In both structures, however, the crystals 146 were grown under non-reducing conditions. The reasons for the two distinct forms of the Cys51 residue are not clear. To further complicate matters, all kinetic measurements of this mutant were made in the presence of 2 —mercaptoethanol to prevent the oxidation of free sulfhydryl groups. It is interesting to speculate that the TC51 mutant enhances the binding of ATP (unlike TA51 and TP51), at least in part, by new interactions arising from the disufide bond. The existence of a reduced form of the TC51 mutant, though, could also indicate that this is the predominant form of the enzyme in the presence of reducing agents. A more detailed discussion of the functional properties of this mutant with regard to this 'structural paradox' may be found elsewhere (M. Fothergill, in preparation). 5.2.4 Solvent Accessibility Calculations In an effort to assess the effect of the position 51 mutants upon the local shape and enviroment in the TyrTS a series of solvent accessibility calculations were carried out and compared with those for the ligand—free wild—type enzyme. Using the program SURFACE (P. Brick and T. Skarzynski, personal communication) the entire solvent—accessible surface (Lee and Richards, 1971) for each mutant coordinate set (derived from simulated substitutions using the program FRODO) was generated by a Connolly—like algorithm (Connolly, 1983). The results are summarized in Table 5.6 which lists the residues where most of the solvent surface—accessibility changes occurred. In the tyrosinyl adenylate complex (Monteilhet and Blow, 1978), the adenine ring lies close to the threonine 51 sidechain and is in van der Waals' contact with the Ca and carbonyl oxygen atoms of Gly 47. It encloses a small vacant void containing the Ala50 residue, too small to accommodate a water molecule (see Figure 5.7a). In the TA51 mutant, the polar hydroxyl group of the threonine sidechain is lost and the void is slightly more open. The presence of an apolar sulfhydryl group in the TC51 mutant does not significantly change in the shape of the void. In the TP51 mutant, however, the void has a greatly reduced volume (see Figure 5.7b) and the local hydrophobicity of the mutated region has increased because of the increased solvent—accessible surface V Table 5.6 Surface accessibilities of wild—type TyrTS and position 51 mutants surface accessibility (») re s id u e TS51 TG51 T h rS l1 TA51 TC512 TP51 TV513 Gly47 N 1 1 1 — 1 1 1 Ca 14 15 14 13 13 13 14 C 1 2 2 1 1 1 2 0 33 32 29 35 26 30 29 His48 Ca — 3 1 2 1 — 2 N51 11 12 12 8 CS2 134 7 12 Cel 17 17 21 17 34 22 21 Ne2 2 1 4 4 154 3 4 Ala50 Ca — 1 — — 1 —— C0 9 13 6 6 6 4 6 Res51 SER GLY THR ALA CYS PRO VAL o r 2 N 4 Cy2 8 N 1 Sy 16 cy 12 Cy2 12 03 12 Ca 7 Oyl 5 03 12 C 6 2 C81 3 In this surface calculation the radius of the solvent extended surface was set to 1.8A to examine accessible regions to apolar groups. 1 Wild—type TyrTS. Calculations were made using coordinates of the reduced form of the TC51 mutant kindly made available by Michael Fothergill. ^Discussed in section 5.3. * \ 2 of residue histidine 48 is rotated approximately 180° in the TS51 structure. 148 Figure 5.7 Slices of van der Waals' envelopes of the wild—type TyrTS and the TP51 mutant structures a. Slices through van der Waals' envelopes containing residues 45—55, 190—193, and the adenosine moiety of the tyrosyl adenylate labelled 321. The 'void', indicated by an arrow, is the empty volume enclosed by the side chain of Thr51 and mainchain components from Leu49, His48 and Gly47. b. The same as a except with proline substituted for theonine at position 51. In the TP51 mutant structure the Cy and C5 atoms of the pyrrolidine ring occlude a large percentage of the original volume of the void. (Pictures produced by a computer program by A.M. Lesk and K.D. Hartman, 1982). 149 of the C7 and C 5 atoms of the proline residue. In the TG51 and the TS51 mutants, the loss of the hydrophobic methyl group of the threonine 51 sidechain creates a larger void. In general, the amount of solvent—accessible surface which is altered for all the mutants is small, approximately 3—5A^, which can be estimated to correspond to a O.lkcal/mole change in hydrophobic free energy based on a correlation relating surface accessible area to empirically measured hydrophobicities for various amino acid residues (Chothia, 1974; Lee and Richards, 1971; Nozaki and Tanford, 1971). The changing shape of the void described in the previous paragraph appears to serve as a marker to indicate the relative bulkiness of the residue at position 51. There appears to be no correlation between void size and activity and only in the most catalytically efficient mutant TP51 is the void completely absent. The results from this calculation do indicate, though, that there may be a correlation between the activities of the various mutants and the apolar nature of the residue substituted at position 51. Specifically, the TP51, TC51 and TA51 mutants exhibit the most 'hydrophobic' surface and are the most catalytically efficient versions of the TyrTS for activation of tyrosine. These mutants are more apolar in nature compared to the wild—type threonine 51 residue which contains the additional polar hydroxyl group. The absence of a side chain entirely in the TG51 mutant produces an enzyme with a decreased level of activity. The effective removal of the apolar methyl group from threonine with respect to the TS51 mutant results in an enzyme with even poorer catalytic activity stressing that the absence of any group at position 51 is less destabilizing than the presence of a residue purely polar in nature. If the correlation between the hydrophobicity and activity is valid then the enhanced catalytic efficiencies observed in the TA51, TC51 and TP51 mutants may be in part due to an incremental improvement in a van der Waals'—type of interaction or favorable dispersion energies (Fersht et al., 1985) between the enzyme and complexes containing ATP and/or the tyrosyl adenylate. 150 5.2.5 Discussion Site—directed mutagenesis of position 51 in the TyrTS has produced the only mutants of this enzyme which display enhanced catalytic properties for the formation of tyrosyl adenylate. Given the vast amount of structural and kinetic data available, it is still difficult to precisely define the origin of the enhanced effects. The structural studies presented here have shown that 'dissection' of the wild—type threonine into its component parts through the construction of mutants with smaller amino acids at position 51 (Ala, Ser and Gly) produces enzymes with structures which demonstrate additional features other than the simple removal of a side chain. This indicates that the contributions of individual groups at position 51 to binding energy as determined through a series of systematic comparisons by Fersht et al. (1985) is not particularly valid. However, comparison of structural and kinetic data in systematic sense with regard to the nature of each residue can provide a powerful means for elucidating the origins of the altered activities of each mutant. The next section describes the design and testing of a theory which aims to determine the significance of the potential hydrophobic interactions which may be responsible for the enhanced catalytic properties of the TP51 mutant, for example, where the crystal structure provides little information about the origins of its effects. It must also be pointed out that the structures of the mutants could also change when substrates bind and react. It is unfortunate that despite numerous attempts to obtain a structure of the TP51 with bound tyrosyl adenylate none was successful. However, as was previously mentioned in section 5.2 the similarity of the structure of the tyrosyl adenylate in the TC51 mutant with the tyrosinyl adenylate in the YRSI structure does provide some validity for the use of the coordinates of this inhibitor in modelling studies to examine the possible effects of each mutation. It is also possible that additional information regarding the interaction of each side chain at position 51 with tyrosyl adenylate may be obtained by using tbese^data in conjunction with molecular dyanamic simulations although the feasibilty of such studies in unknown at present. In general, though, the crystallographic studies have provided an initial model from which one can make preliminary assessments about the function of. particular side 151 chains in an active site. This information in combination with kinetic data may then allow one to predictably alter the catalytic function of an enzyme. 5.3 The Thr51 — >Val Mutation: Testing 'Hydrophobicity' 5.3.1 Design of the Mutation As discussed in section 5.2.5 one possible theory which could partly explain the observed enhanced activity of the TP51 mutant could be the increased apolar surface exhibited by the proline in comparison to the wild—type threonine. The destabilizing effect of the Thr51 polar hydroxyl group has been described in detail in previous kinetic studies (Wilkinson et al.t 1984; Fersht et al., 1985). The question as to why the proline substitution produced a more catalytically efficient enzyme in comparison to alanine and cysteine was never really addressed once the crystal structure of the TP51 mutation was determined and indicated no structural change (Brown et al., 1986; Brown et al., 1987). It is not entirely obvious that the change from an alanine to cysteine necessarily indicated an enhanced activity due to increased apolar surface area. Both Fersht et al. (1985) and Ho and Fersht (1986) suggested that the sulfhydryl group of the cysteine might really be making an improved hydrogen bonding interaction with the ribose ring oxygen of the tyrosyl adenylate. The possible participation of this residue in a disulfide bridge (M. Fothergill, in preparation), though, makes the existence of this polar interaction less likely. In order to test the significance of increased apolar surface as related to possible improved van der Waals' interactions it was necessary to design a mutant whose activity could be related to this property exclusively; hence followed the replacement of threonine 51 with a valine (TV51). The crystal structures described in section 5.2.4 suggested that the TV51 mutant should not cause any alteration of the main chain structure in the ligand—free enzyme. The primary change would be an increase in apolar surface resulting from the additional methyl groups when compared to the alanine residue in the TA51 mutant. This is shown in Table 5.6 which gives the solvent—accessible surface of a valine residue modelled at position 51 in the TyrTS. If apolar surface area was not a 152 contributing factor to the observed catalytic enhancements then the TV51 mutant would not be expected to differ significantly from the TA51 mutant. 5.3.2 Kinetic Characterization Kinetic data presented as part of this thesis (sections 3.6 and 3.7) demonstrated that the TV51 displayed the predicted enhanced activity for the formation of tyrosyl adenylate. The 4—fold increase in the rate of adenylate formation combined with a 24—fold increase in its affinity for ATP yielded a 100—fold increase in the specificity constant, which is the largest value obtained for any TyrTS mutant (shown in Table 3.2). The corresponding Gibbs' free energies of complexes of the TV51 mutant for the formation of tyrosyl adenylate are given in Tables 5.4 and 5.5 for comparison. TV51 is similar to the TC51 in that it demonstrates an enhanced (and even greater) affinity for ATP. This observation is further illustrated in the form of the linear free energy relationships described in section 5.2.3 which also shows TV51 departing from linearity in much the same manner as the TC51 mutant during the formation of the E-Tyr-ATP complex (Figure 5.5a). This result suggests that the TV51 may also be employing a different reaction mechanism for the binding of ATP in the pre—transition state. The proximity of the TC51 and TV51 data points implies that the mechanism could be similar. Furthermore, as valine possesses no polar groups which could interact in an improved 'hydrogen bonding interaction' it seems likely that the enhanced activity demonstrated by the TC51 mutant could be, at least in part, the result of improved van der Waals' interactions. This hypothesis treats the exposed S 7 atom of the TC51 mutant (Table 5.6) as apolar in nature rather than assuming it is involved in a polar interaction with ATP/tyrosyl adenylate. It might, on the other hand, be the case that the similarities between the TC51 and TV51 are fortuitous and that two different reaction mechanisms are being employed. This point will be discussed in the following section in the context of crystallographic and modelling studies of position 51 mutants of the TyrTS. 153 5.3.3 Discussion The design of the TV51 mutant is unique among the mutants of the TyrTS in that its aim was to determine the significance of hydrophobic interactions with the substrate. It is also the first mutation which was predicted to enhance the the catalytic properties of the TyrTS based upon a correlation of enzymic activity with the chemical nature of a set of residues at substituted at the same position. The crystal structures to date of the position 51 mutants, however, do not clearly indicate where such interactions would be made with the tyrosyl adenylate although the proximity of residue 51 to the ribose ring makes that moiety the most likely candidate. Even more puzzling is the inability of any structural studies to provide information about the location of unreacted ATP when it is bound to the enzyme although numerous attempts have been made (Monteilhet and Blow, 1978; M. Fothergill, unpublished data). It has been suggested (D.M. Blow, personal communication) that the ATP could be loosely bound in the pocket of the active site and only upon formation of the tyrosyl adenylate does it become ordered. If this occurs then it may be possible that both the TC51 and TV51 mutants could interact more strongly with ATP, especially with the hydrophobic components of the adenine moiety. The kinetic data described in the previous section suggest that the TC51 and TV51 mutants might bind ATP by a different reaction mechanism compared to say the wild—type enzyme. However, modelling studies which place the valine in an orientation most commonly adopted by this residue in an a —helix (x, = —180 in 87% of a —helical structures; survey by McGregor et al., 1987) show that both the Cy2 groups of threonine and valine would nearly superimpose themselves in the TyrTS structure (Figure 5.8a). Even more interesting is the structure of the reduced form of the TC51 mutant which also shows the orientation of its sulfhydryl in the same direction as these Cy2 groups as opposed to the O 7 I group of the threonine (M. Fothergill, in preparation; shown in Figure 5.8b with the modelled TV51 mutant coordinates). It becomes difficult to rationalize how the interaction of the C 72 group of Thr51 and the C72 group of Val51 would differ. What seems more likely is that the O 7 I group of the Thr51 residue in the wild—type structure is destabilizing the binding of ATP to « 154 Figure 5.8 Comparison of the modelled TV51 side chain with other TyrTS structures a. Residues 50—52 of the modelled TV51 (shown by thick lines) and the wild—type (in thin lines) TyrTS structures. The C7 I and O 7 I atoms of the Val51 and Thr51 residues are labelled, respectively. The Cy2 atoms of both residues are denoted with a single label. b. Residues 50—52 of the modelled TV51 (shown by thick lines) and the TC51 (in thin lines; coordinates kindly provided by Michael Fothergill) TyrTS structures. The C7 I and Cy2 atoms of Val51 and the S 7 atom of Cys51 are labelled. c. Residues 50—52 of the modelled TV51 (shown by thick lines) and the TP51 (in thin lines) TyrTS structures. The C7 I and Cy2 atoms of Val51 and the C 7 atom of Pro51 are labelled. 155 the enzyme, maybe by unfavorable lone pair—lone pair interactions similar to those discussed earlier (section 5.2.3), and this effect masks the favorable hydrophobic interactions of its Cy2 group described by Fersht et al. (1985). It follows that the slope of the line (0) in Figure 5.5a (which describes the binding of ATP to the TyrTS), determined by a linear regression of the all data shown, could shift to 0.20 thus implying that as much as 20% of the binding energy available to the TyrTS enzyme is used for binding ATP. If one chooses to accept to altered position of the line in Figure 5.5a then it would also follow that the TV51 and TC51 would probably be using the same hydrophopbic mechanism of interaction proposed in this thesis. When the crystal structure of the TC51 mutant (M. Fothergill, in preparation) is compared with the modelled coordinates of the TV51 mutant it is clear that the respective sulfhydryl group and the Cy2 group occupy approximately the same position (Figure 5.8b) with nearly the same amount of exposed area (see Table 5.6). Furthermore, the location of the free sulfhydryl group in this structure could not form the proposed hydrogen bonding interaction with the ribose ring of the tyrosyl adenylate supposed responsible for its enhanced activity. Recalling the TP51 mutant it might then seem curious that the proline with as much exposed apolar surface as cysteine or valine (Table 5.6) does not demonstrate the same enhanced binding of ATP. Comparison of the TP51 crystal structure with the modelled valine coordinates, Figure 5.5c, shows that although the Pro51 residue introduces new hydrophobic groups compared to Thr51 or Ala51, the location of its C 7 and C5 atoms signifcantly differs from the location C 72 and S 7 groups implicated to interact with ATP. It may also be the case that proline with its 'unusually' placed hydrophobic groups may interact with ATP by a slightly altered mechanism and it is the TP51 mutant whose point on Figure 5.5a which may show some departure from linearity. However, it is also satisfying to observe that the data for the TV51 mutant falls along the same line described by the linear free energy relationships given in Figures 5.5b and c. These graphs show that TV51 is employing the same reaction mechanism for binding the transition state and the tyrosyl adenylate complex as all the other position 51 mutants of the TyrTS which demonstrate 156 enhanced activity. The common feature shared by these mutants is increased hydophobicity relative to the wild—type enzyme. These results support the hypothesized interaction of apolar groups with the tyrosyl adenylate „' 7 ^xyalso suggest that the location of apolar groups at position 51 does kc4t vuM iv bcso restricted in order to produce the enhanced catalytic/binding effect. It must also be noted that the accurracy of the pre—steady state kinetic data for the pyrophosphorolysis reaction fell outside typical standard error ranges. Because these errors and other inconsistencies discussed in section 3.6.3 it has been recommended that data collection be repeated if possible to obtain more reliable values for k — 3 and Kpp. A complete error analysis of this data with regard to the LFER's presented in this section will not be attempted here. However, some confidence in the data may be derived from the fact the data point for TV51 only departs from linearity in the LFER which describes the change in binding energy associated with the formation of the E-Tyr-ATP complex. In this case the position of TV51 along the x—axis (which is influenced by k —3 and Kpp) is reasonable in that it appears near other position 51 mutants which demonstrate enhanced stabilization of the E-Tyr—AMP complex. Its position along the y—axis (which may now appear suspicious) is strictly dependent upon its value for the dissociation constant of ATP (K^ *) which was accurately determined in the forward reaction. However, as mentioned earlier, confidence in all these values can be best achieved by a redetermination of the rate and equilbruim constants in question. 5.4 The Ala50-»Pro Mutation: Prolines in Helices 5.4.1 Design of the Mutation Cantor and Schimmel (1980) point out that the primary cause for proline to disrupt an a —helical structure is not because the proline nitrogen cannot participate in a hydrogen bond. They suggest that steric conflicts involving the atoms of the pyrrolidine ring restrict rotational freedom of the preceding residue thereby preventing it from adopting an a —helical conformation. In the wild—type structure of the TyrTS * 157 alanine is present at position 50 which is identical to the case Schimmel and Flory (1968) used to demonstrate how the conformation accessible to a residue preceding proline on a polypeptide chain is limited. Figures 5.9a and b show the energy contour diagrams for an L—alanyl residue in a general polypeptide chain (Brant et al., 1967) and in the case where it is specifically succeeded by a proline (Schimmel and Flory, 1968). In the latter study conformational angles in the region of the plot which describe a —helical structures ( —18 0 ^ ^ —40, —10 ^ ^ —60; Ramachandran et al., 1963) were considered inaccessible. In the refined structures of the wild—type TyrTS (ligand—free or containing the inhibitor tyrosinyl adenylate) where the alanyl residue is succeeded by a threonine in an a —helical region, the angles of Ala50 fall within the previously described a —helical conformational limits (Brick, Bhat and Blow, in preparation; Monteilhet and Blow, 1978). However, as was described in section 5.1.4 the presence of the proline at position 51 in the TP51 mutant displayed effectively no mainchain or side chain distortions thereby maintaining the a —helical conformational angles of the Ala50 residue. A comparative listing of the 45—55 in the wild—type TyrTS and TP51 mutant structures is shown in Table 5.7. The apparent discrepancy between the theoretically and experimentally derived angles is not unique. A search of the Birkbeck Relational Database 'ORACLE' (courtesy of Dr. Michael Sternberg) yielded at least nine protein structures at a resolution better than 2.5A, including deoxy—myoglobin and subtilisin BPN, which contained a residue in an a —helical structure exibiting allowed conformational angles despite the fact that it was succeeded by a proline. Cantor and Schimmel (1980) mention some of the factors which can cause this discrepancy between the theoretically and experimentally determined values for rotation angles. Included is the fact that the energy contour diagrams are calculated assuming fixed bond angles and bond lengths and that variations of a few degrees may change the shape of the 'allowed' regions. They also point out that the angles which are experimentally determined are also subject to errors of at least several degrees. Specifically, this section addresses another question: whether there are intrinsic properties associated with the presence of a proline in the a —helical portion of the t l, 1967). al., et a. ciml n Foy 1968). Flory, and Schimmel b. Energy contour diagram for an L—alanyl residue in a polypeptide chain (after Brant Brant (after chain polypeptide a in residue L—alanyl an for diagram contour Energy Energy contour diagram for an L—alanyl residue that is succeeded by proline (after (after proline by succeeded is that residue L—alanyl an for diagram contour Energy ^i(dcg) Figure 5.9 Energy contour diagrams for an L—alanyl residue residue L—alanyl an for diagrams contour Energy 5.9 Figure -180 i. tkn rm atr n Shme, 1980) Schimmel, and Cantor from (taken Table 5.7 The angles of residues 45—55 in the wild—type TyrTS and TP51 mutant crystal structures w ild -ty p e TP51 re s id u e Phi P si Phi Ps i His45 -140 -179 -141 179 I le46 -63 -14 -60 -13 Gly47 -71 -24 -72 -20 His48 -95 -8 -96 -12 Leu49 -63 -38 -61 -42 Ala50 -63 -52 -57 -55 Thr51 -63 -42 Pro51 -59 -44 I le52 -56 -54 -59 -56 Leu53 -69 -31 -65 -33 Thr54 -64 -38 -65 -35 Met55 -58 -45 -59 -48 Arg56 -57 -44 -55 -46 Arg57 -65 -40 -63 -41 Phe58 -67 -46 -66 -44 Gln59 -60 -39 -61 -44 Gln60 -63 -29 -60 -30 160 45/55 segment of the TyrTS structure which may possibly contribute to the enhanced activity of the TP51 mutant. Proline does not appear to have any effect upon the conformational angles of the adjacent residues. A comparative listing of B—factors in Table 4.5 gives no evidence for any localized changes on the ordering of the residues in this helical structure. There is no indication in the current structural data which suggest that Pro51 exerts a conformational constraint on this local structure. The design of another proline containing mutant had two aims. The first was to place a proline in the 45/55 segment near position 51 which would, in theory, make no direct interaction with any of the reactants or products for the adenylation of tyrosine. The assessment of the effects of this mutation upon catalysis could be carried out using the traditional kinetic assays of the TyrTS enzyme. It was also necesssary to ensure that such a mutation would not cause any additional structural effects as shown in the crystal structure of the TP51 mutant. Hence, knowledge about the 'allowability' of prolines in a —helical structures gave an expectation that a new proline could be placed in the helical portion of the 45/55 segment without introducing mainchain structural changes. Using the amino acid substitution facility in the molecular modelling program FRODO (Jones, 1978), a proline was systematically replaced at each position which displayed 55; Figure 5.3). The coordinates of each modelled mutant were displayed using an Evans and Sutherland PS300 molecular graphics system. Because the aim of the mutation was to cause no structural movement no torsion angles were adjusted. Instead, for each new proline residue all contacts less that 4A from the side chain atoms were determined. Placement of the proline at position 50, the Ala50-»Pro mutation, proved to be most suitable. A structural comparison with the Thr51-*Pro mutation revealed that the starting angles were very similar: ( —62, —42) for residue 51 and ( —63, —52) for residue 50. The conformational energy versus \J/ for an isolated proline within a polypeptide chain has two minima at ^=—55° and ^=145° (Schimmel and Flory, 1968). Because —52° at position 50 was even closer to a calculated energetic mininum than that for Thr51 before mutation, introduction of 161 proline at position 50 was expected to have very little effect upon the surrounding structure. In addition, the contact distances for the side chain atoms of Pro50 appeared nearly identical to those for the Pro51 residue accounting for the (i — 1) shift along the polypeptide chain. The relevant comparison is shown in Table 5.8. 5.4.2 Discussion Since the construction of this mutant, Matthews et al. (1987) have described a strategy for enhancing protein thermostability which includes the replacement of selected amino acids in a given protein by proline. As discussed in section 1.2.2C, because the pyrollidine ring of proline restricts the conformations of this residue compared to other amino acids, it is hypothesized that its presence in a polypeptide backbone may reduce the configurational entropy of unfolding of a protein. The objective of their mutational design though, was the same as that for the AP50 mutant: to choose substitution sites that would cause minimal perturbation of the three-dimensional structure of the given protein. In order to assess the compatibility of a site with this type of substitution, they made a survey of structures to determine the range of conformational angles of prolines that occur in actual proteins. However, the restrictions placed upon the angles of the residue preceding a proline residue by Schimmel and Flory (1968) were strictly adhered to by Matthews et al. (1987). As was demonstrated in the previous section, these restrictions do not necessarily describe what is actually observed in some protein crystal structures and hence indicates that the population of potential substitution sites is probably greater than Matthews et al. (1987) have assumed. In the case of their proline mutant, the design was successful. The crystal structure of the mutant displayed no structural changes and the new protein demonstrated enhanced thermal stability (higher melting temperature). The AP50 mutant of the TyrTS has been crystallized in the trigonal P3j21 form (S. Onesti, personal communication) exhibited by all mutants whose structures have been determined as part of this thesis, but diffraction data has yet to be collected. No formal kinetic studies have been attempted to date but the AP50 mutant is known to be active for the formation of tyrosyl adenylate based upon a simple—time course 162 Table 5.8 Contact distances for proline in the TP51 and AP50 TyrTS mutants Pro51: CS Pro50: CS 0 0 Atom Distance(A) Atom Distance(A) 050 3.61 049 3.58 C50 2.50 C49 2.47 Ccx50 2.98 0x49 2.94 N50 2.92 N49 2.83 N52 3.65 N51 3.63 048 3.24 049 3.63 C48 3.62 C47 3.98 C49 3.93 C48 3.40 049 3.93 048 3.94 C/350 3.17 C/349 3.28 Pro51: cy Pro50: C7 0 0 Atom Distance(A) Atom Distance(A) C50 3.67 C49 3.62 N52 3.78 N51 3.77 047 3.42 048 >4.00 The contact distances shown are measured from the C6 or Cy group of each proline residue to the 'Atom' listed below. The distances measured from the Pro51 residue are taken from the coordinates of the TP51 mutant crystal structure. The distances measured from the Pro50 residue were made using coordinates modelled into the ligand—free wild—type crystal as described in section 5.4.1. 163 examination of activity using active site titration (section 3.4). 5.5 The His48-*Gly Mutation 5.5.1 Kinetic Characterization The N$1 group of the histidine side chain at position 48 (His48) has also been identified to be involved in a possible polar interaction with the ribosyl ring oxygen ( 0 —4) of tyrosinyl adenylate in the original wild—type crystal structure (Monteilhet and Blow, 1978; Bhat et al., 1982: Bhat, Brick and Blow, in preparation). Deletion of the entire side chain in the His48-»Gly mutation resulted in a 1.2kcal/mole destabilization of the transition state as measured by pyrophosphate exchange kinetics (Table 5.9; Carter et al., 1984). The free energy profile and accompanying difference energy diagram, Figure 5.10, shows that the glycine substitution caused a small change in the dissociation constant for tyrosine (AT^) and a net destabilization of 0.44kcal/mole in the E-Tyr-ATP complex, 1,24kcal/mole in the transition state and 1.47kcal/mole in E-Tyr—AMP (Wells and Fersht, 1986). Studies by Lowe et al. (1985) suggested that the slight increase in Kt, typical of position 48 mutants, may simply reflect the loss of an electrostatic interaction with the carboxylate of the tyrosine substrate. The pyrophosphate exchange reactions for double mutants containing the His48->Gly mutation, namely the HG35HG48 and HG48TP51 mutants, are also included in Table 5.9. Both mutants demonstrate a destabilization of the transition state, 2.3 and l.lkcal/mole respectively. The significance of these values with respect to the single mutations from which each double mutant is composed has been discussed in detail in section 5.1.3. 5.5.2 The Hydrolysis of Tyrosyl Adenylate Wells (1987) examined the rate of decay of enzyme bound tyrosyl adenylate due to hydrolysis for a set of TyrTS mutants as described by the following reaction: E-Tyr-AMP + OH- J E + Tyr + AMP. 164 Table 5.9 Pyrophosphate exchange activity wild—type and mutant (position 48) TyrTS’s Enzyme k c a t Km(ATP> ^cat/^M ( s ' 1 ) ( mM) (s-lM ”1 ) TyrTS(wi Id-type)3 8 .3 5 1.08 7730 HG482 1 .9 1.3 1460 CG353 2.8 2.6 1120 CG35HG482 1 .3 2 8.0 177 TP514 12.0 0.058 208000 HG48TP512 2 .9 2.1 1380 ^Fersht et al., 1985. ^Carter et al., 1984. ^Winter et al., 1982. 4 H o and Fersht, 1986 Figure 5.10 Gibbs' free energies of enzyme—bound complexes of the HG48 mutant (taken from Wells and Fersht, 1986) 8.0 - CE.T-A]* e.o- 4.0- 2.0 - 0.0- - 2. 0 - -4.0- - 8 .0 - - 8. 0- - 10. 0 - G k c a l m o l’ ’ His-Gly48 a. The Gibbs' free energy profile for the formation of tyrosyl adenylate and pyrophosphate, as defined by Scheme I (section 3.6.1), by wild—type (energy levels in broken lines) and the HG48 mutant (energy levels in heavy lines) TyrTS's, using standard states of 1 M for tyrosine, ATP and pyrophosphate. His-Gly48 3.0-i 2.0 - , , Ui I - i w kcal mol”’ ui h b. The Gibbs' free energies of enzyme—bound complexes of the HG48 mutant TyrTS relative to those of the wild-type enzyme. The linear free energy relationship between tyrosyl adenylate hydrolysis rate constant and dissociation constant, shown in Figure 5.11, demonstrated that for most mutants 20% of the E*Tyr—AMP binding energy was released in the transition state. He noted that this value was very similar to that obtained for the pyrophosphorolysis of the E*Tyr—AMP complex. This was interpreted to mean that both hydrolysis and pyrophorolysis in the TyrTS have a geometrically similar transition state involving the formation of a pentacoordinate structure at the a —phosphorus. Specifically, hydrolysis would involve an S^2 attack by the OH- group on the a-phosphorus resulting in the loss of tyrosine. In the case of the HG48 mutant, the complete removal of the histidine side chain resulted in an enhanced effect on the rate of hydrolysis. Further analysis indicated that this mutant appeared to have access to a lower enthalpy, lower entropy hydrolysis pathway compared to the other mutants examined in this study. This is illustrated by the deviation of the HG48 mutant from the linear regression of the LFER hydrolysis data in Figure 5.11. The HA48 (His48-»AIa) mutant, which also demonstrates an enhanced rate of hydrolysis (but not as great as the HG48 mutant), deviates in this plot as well. In the proposed mechanism the presence of a large residue such as histidine or asparagine was viewed as sterically blocking off this alternative route of hydrolysis. Wells supported this interpretation by pointing out that in the wild—type structure the His48 residue lies on the line of attack that an OH~ species would have to take in order to produce a pentacoordinate transition state. In addition, the similarity of the kinetic data on the HG48 and HA48 mutants seemed to suggest that the elevated rate of hydrolysis was not exclusively dependent upon the complete removal of the histidine side chain. This observation, however, did not necessarily preclude the possibility of a structurally related mechanistic alteration. 5.5.3 The Crystal Structure The crystal structure of the HG48 displayed movements of the mainchain structure Figure 5.11 The linear free energy relationship between the tyrosyl adenylate hydrolysis rate constant (fcjj) and dissociation equilibrium constant (K^) (taken from Wells, 1987) associated with the complete removal of the histidine at this position. The movements propagated in the direction of the carboxyl terminus affecting, in particular, the orientation of the Thr51 side chain. When the double mutant test was applied to the Thr51-»Pro mutation in conjunction with the the His48-*Gly mutation a coupling energy of 1.9kcal/mol was observed, indicative of a structurally mediated interaction between these two positions (Carter et al., 1984). The original interpretation of this result was that the proline at position 51 was distorting the a —helix in some mannner so as to affect the interaction of His48 with the tyrosyl adenylate. The crystal structure of the ligand—free TP51 mutant (section 5.1.4) provided no evidence of a mainchain or a side chain distortion at position 48 which would easily explain the kinetic data. However, the crystal structure of the HG48 mutant displayed mainchain and side chain movements throughout the 45/55 segment which as a consequence affected the orientation of the Thr51 side chain. In comparison with the ligand—free wild—type structure (Figure 4.7h) Xi f°r Thr51 has rotated from 65° to 87° in the HG48 crystal structure. This movment shifts the position of its Cy2 and O7I groups 0.2X and 0.7X, repectively. The temperature factors of these groups as well as the rest of the 45/55 segment have also increased (Table 5.5) indicative of a general increase in disorder. The position of Thr51 has also shifted relative to its position in the YRSI structure. A 1.4A shift of its O7I group places it 3.9X from the 0 —4 group of the tyrosinyl adeylate (compared to 3.6X in the YRSI structure). In general such movements would be expected to affect the interaction of the Thr51 residue with tyrosyl adenylate. It is also possible that when proline is present at position 51 in the HG48TP51 mutant that the mainchain movements resulting from the removal of histidine at position 48 may also affect its orientation and interaction with tyrosyl adenylate. With regard to the kinetic data on the CG35HG48 mutant, the movements associated with the HG48 mutant do not appear to affect the position of residue Cys35. In addition, the structure of the CG35 mutant (M. Fothergill, in preparation) has also shown that removal of the cysteine side chain at position 35, although affecting the orientation of Thr51 does not shift the position of His48. Both structures (HG48 and CG35) appear consistent with the kinetic data which detected no coupling energy between these two « 169 mutations and hence no structural interaction (Carter et al., 1984). The structure of the HG48 mutant then has given the rather unexpected result demonstrating a structural interaction between the positions 48 and 51. However it is dificult to know if these movements alone could account for the 1.9kcal/mol coupling energy which the double mutant test has detected. It has been noted that HG48 and HG48TP51 have nearly the same activity. Lowe et al. (1985) interpreted this in conjunction with their results on the His48-»Asn and His48-*Gln mutants, which demonstrated improved activity when Thr51 was also mutated to Pro, to mean that the presence of a hydrogen—bond doner at position 51 was necessary to bring about an improvement in transition state stabilization. The results of the original double mutant test with HG48 and TP51 may also be restated to say that the effect of placing glycine at position 48 may be so destabilizing to the transition state that it cannot even be compensated by placing proline at position 51. In the wild—type crystal structure the histidine side chain of position 48 forms part of the exposed entrance to the active site (Figure 5.12a). Examination of the HG48 crystal structure shows that the removal of histidine enlarges this entrance (Figure 5.12b). This increases the solvent—accessible surface of residue 51 as well as all other neighboring residues exposed in the 45/55 segment and allows a new water molecule, HOH 604, to occupy the space previously filled by the His48 side chain which will not necessarily be displaced when tyrosyl adenylate binds. In addition, the increased size of the entrance to the active site also suggests that bound tyrosyl adenylate will also be more exposed and susceptible to displacement by solvent, possibly to the extent that the enhanced binding properties of the TP51 mutation are ineffective. It would also follow that the HN48 and HQ48 mutations, while still providing a hydrogen—bonding donor, may also be protecting the active site against the destabilizing effects of competing solvent thereby allowing the TP51 mutant to exercise its enhancing catalytic effects. With regard to the hydrolysis reaction discussed in the previous section Wells (1987) described that His48 lies in the line of attack that an OH “ species would have to take in order to produce the pentacoordinate transition state structure implicated to exist from his kinetic studies of this reaction. In the HG48 crystal structure the Figure 5.12 Van der Waals' radii of the entrance to the active site of the TyrTS a. The van der Waals' radii of residues near His48 located at the entrance to the active site in the YRSI crystal structure. Histidine 48 (His) and the atoms of tyrosinyl adenylate (Inh) are emphasized with a dark border. b. The van der Waals' radii of residues near Gly48 located at the entrance to the active site in the HG48 crystal structure. Glycine 48 (Gly) and the atoms of tyrosinyl adenylate (Inh; coordinates from the YRSI crystal structure) are emphasized with a dark border. t 171 histidine side chain has been replaced by a new solvent molecule, HOH 604. This water molecule approaches hydrogen—bonding distance (3.4A) of the amino group of residue 45 and has a reasonably low temperature factor (24.9X2) for this structure. When tyrosyl adenylate is bound to the enzyme HOH 604 lies in a direct line of attack to its a —phosphorus group. This is shown in Figure 5.13a using the coordinates of the tyrosinyl adenylate which place HOH 604 5.9X from the a —phosphorus group. The HA48 mutant also displayed an enhanced level of hydrolysis, although slightly decreased with respect to the HG48 mutant. Figures 5.14a and b show that alanine, modelled at position 48, can still leave enough space in the pocket previously occupied by the histidine side chain to accomodate HOH 604. The close proximity of these two groups, however, suggests that HOH 604 may experience some steric hinderance with respect to its line of attack on the a —phosphorus group of the adenylate (see Figure 5.13b). In addition, the C0 group of Ala48 probably displaces HOH 604 to some extent as well thereby lowering its occupancy (concentration) and subsequently the hydrolytic activity of HA48. Alanine at position 48 also enlarges the entrance to the active site with respect to the original wild—type structure. The possibility of increased solvent attack on the adenylate as related to the increased accessibilities in the HA48 mutant is also reasonable. It cannot be said whether the mainchain and side chain movements shown in the HG48 crystal strutcure play any definitive role in enhancing hydrolysis or whether ther would be any similar structural changes associated with the HA48 mutation. However, crystal structures of the TG51 and TA51 mutants have shown that the structural changes affecting residues 48—51 incurred by placing glycine at position 51 were not demonstrated when alanine was placed at that position. This result gives some support to the hypothesis that the similar effects of HA48 on catalysis may not necessarily be related to mainchain distortion of the enzyme. 5.5.4 Discussion The structure of the HG48 mutant has served as a model to study the strucural basis of many proposed interactions and activities. The interpretation of the activity of this mutant for the formation of tyrosyl adenylate with respect to the double mutant -* 172 Figure 5.13 Model for the hydrolysis of the tyrosyl adenylate in the HG48 and IIA48 mutants a. The in line nucleophilic attack (dotted line) of water molecule HOH 604 on the a —phosphorus of the tyrosyl adenylate (shown in blue using coordinates of the tyrosinyl adenylate) in the HG48 crystal structure. Glycine 48 is shown in red and HOH 604 is shown participating in a hydrogen bond (dotted line) with the amino group of residue 45. b. The same as a with alanine (shown in red) at position 48. 173 Figure 5.14 Van der Waals' radii of the active site of TyrTS a. Van der waals' radii of the active site of the HG48 crystal structure shown at a view perpendicular to Figure 5.13. Glycine 48 (Giy) and the atoms of tyrosinyl adenylate (Inh) are emphasized with a dark border. Water molecule HOH 604 is shown by two concentric circles. b. The same as a with alanine (Ala) modelled at position 48 (emphasized with a dark border). ♦ 174 studies is complex. Given both the the TP51 and HG48 mutant structures it is difficult to deduce how the removal of the histidine, in particular, affects the activity of proline at position 51. The crystal structure of the HG48TP51 mutant has been determined (S. Onesti, personal communication) and demonstrated some mainchain movement associated with the HG48; however, heterogeneity at position 51 (reasons unknown) has made it difficult to determine whether such mainchain changes have altered the position of the proline. side chain. The redetermination of the HG48TP51 crystal structure using protein grown from a pure phage stock may be necessary in order to properly interpret its catalytic properties. With regard to the hydrolysis reaction, existence of HOH 604 has provided a structural model consistent with the kinetic evidence which aims to describe a mechanism for the enhanced level of hydrolysis of the HG48 mutant. Modelling studies of the HA48 mutant have also provided a model consistent with kinetic data to explain its activity. However, the mechanism which explains enhanced catalysis assumes that nucleophilic attack is at the a —phosphorus of the tyrosyl adenylate. The attack may occur at the tyrosine carboxyl group or possibly at both this group and the a —phosphorus group. If this were the case then the similarites between the hydroylsis and pyrophosphorolysis LFER's (which suggest a similar transition state structure for both reactions) may be fortuitous (Wells.1987). However, it is interesting to note that increased accessibilties of side chains on the enzyme as related to the removal of histidine at position 48 do not extend beyond the region of the structure which interacts with the adenosine moiety of the tyrosyl adenylate. Additional investigation of the hydrolysis mechanism may rely on proposed studies which would use l^CXHjO) to label the site of attack (Wells, 1987). 5.6 Concluding Remarks It is probably quite reasonable to say that the interpretation of the structural changes associated with a single site mutation can be very simple in some cases and very complex in others. However, interpretation of such structure with regard to their functional properties is never simple. The original aim of this study was to determine f 175 the structure of a single mutant in order to explain its altered activity. This aim, however, could not be achieved without information gained from a systematic study of other mutants at the same position. Furthermore, from this study it was possible to design another mutant whose properties could be successfully predicted. Since prediction may be considered the ultimate aim of a protein engineer, the work presented here gives hope that such a goal can be achieved. APPENDIX I. Materials BUFFERS and DYES 1 X TE. pH8 lOmM Tris-HCl, 0.1 mM EDTA 1 X TM. pH8 lOmM Tris-HCl, lOmM MgCl2 kinase buffer. pH8 50mM Tris-HCl, lOmM MgCl2 1 X TBE. pH8.3 lOmM Tris, lOmM Borate, 2.5mM EDTA 6 X SSC. PH7.2 0.9M NaCl, 0.009M sodium citrate, 0.006M EDTA hybridization Buffer 0.9M NaCl, 0.09M Tris-HCL pH7.4, 0.006M EDTA, 0.5% NP40, 0.2% SDS, 2 X Denhardt's Solution; Nalgene filter the solution and store at — 20°C. 100 X Denhardt's Solution 2% bovine serum albumin (nuclease free grade), 2% polyvinyl—pyrrolidine, 2% ficoll 400; Filter, sterilize and store at — 20°C. protein sample buffer lOmM Tris/Biscine pH8.3, 0.5M 2—ME, 25% glycerol, 2% SDS, 0.02% bromophenol blue formamide dves 95% formamide, 0.3% bromophenol blue, 0.3% xylene cyanol FF, lOmM EDTA PLATES and AGAR Minimal aear + glucose plates constituent concent rat ion k 2hpo 4 5.25 kh 2po 4 2.25 (nh 4 )S04 0 .5 MgS04 0.1 sodium citrate 0.25 agar 7.5 glucose 1.0 v itam in B1 0.0025 H dates const i tu en t concent rat ion bactotryptone 1.0 NaCl 0.8 agar 1.5 H too agar const i tuent concentration bactotryptone 1.0 NaCl 0 .8 178 Appendix II. Refined Coordinates and Temperature Factors of New Atoms in Mutant Crystal Structures TP51 N PRO A 51 -22.332 19.109 -4.943 5.05 CG PRO A 51 -20.366 17.834 -5.263 8 .1 2 CD PRO A 51 -21.648 18.278 -5.930 6.56 CB PRO A 51 -20.116 18.746 -4.112 8.63 CA PRO A 51 -21.451 19.436 -3.838 8.61 C PRO A 51 -21.294 20.940 -3.715 9.53 O PRO A 51 -21.315 21.426 -2.569 14.15 0 HOH A 601 -17.736 15.182 -8.847 32.18 0 HOH A 602 -13.452 5.300 -15.746 22.64 O HOH A 611 -12.099 14.343 -6.333 19.91 0 HOH A 612 -15.083 10.093 -9.926 34.06 O HOH A 613 -3.998 29.805 -1.636 32.22 O HOH A 614 -42.893 11.547 - 1 2 .8 6 8 29.25 O HOH A 615 -30.018 8.212 2.685 32.68 TA51 N ALA A 51 -22.404 19.148 -5.240 5.24 CB ALA A 51 -20.122 18.748 -4.473 5.73 CA ALA A 51 -21.442 19.385 -4.160 7.28 C ALA A 51 -21.333 20.883 -3.894 10.82 0 ALA A 51 -21.432 21.325 -2.730 12.99 N TYA 321 -11.303 13.515 -3.918 19.64 OH TYA 321 -8.622 17.326 0.388 9.36 CD2 TYA 321 -9.059 16.677 -3.197 10.65 CE2 TYA 321 -8.451 17.021 -1.994 10.97 CZ TYA 321 -9.179 16.997 -0.811 12.06 CE1 TYA 321 -10.518 16.622 -0.834 14.70 CD1 TYA 321 - 1 1 .1 0 2 16.244 -2.058 14.67 CG TYA 321 -10.398 16.275 -3.251 11.96 CB TYA 321 -11.088 15.900 -4.522 13.80 CA TYA 321 -11.972 14.655 -4.578 15.30 C TYA 321 -12.230 14.250 -6.033 16.84 01 TYA 321 -13.213 14.737 -6.625 17.43 02 TYA 321 -11.431 13.469 -6.564 17.43 179 TG51 N GLY A 51 - 2 1 787 19.123 -5.104 10.46 CA GLY A 51 -2 0 891 19.558 -4.042 13.21 C GLY A 51 -2 0 812 21.056 -3.878 15.35 0 GLY A 51 -2 0 621 21.556 -2.739 19.82 N TYA 321 - 1 0 522 13.611 -4.301 18.23 OH TYA 321 - 8 619 17.398 0.323 16.82 CD2 TYA 321 -9 014 16.770 -3.253 14.60 CE2 TYA 321 - 8 418 1 7 .1 1 1 -2.041 15.54 CZ TYA 321 -9 174 17.058 -0.878 16.09 CE1 TYA 321 - 1 0 499 16.661 -0.926 17.45 CD1 TYA 321 - 1 1 062 16.295 -2.148 18.05 CG TYA 321 -1 0 334 16.356 -3.336 14.94 CB TYA 321 - 1 1 007 15.985 -4.625 14.25 CA TYA 321 - 1 1 566 14.567 -4.687 16.26 C TYA 321 -1 2 057 14.257 -6.104 18.21 01 TYA 321 -13 132 14.820 -6.407 18.17 02 TYA 321 -1 1 399 13.511 -6.848 18.08 0 HOH A 601 -17 344 15.012 -8.975 30.93 0 HOH A 602 -13 471 5.380 -15.633 27.19 0 HOH A 603 -17 942 31.061 -17.976 32.16 TS51 N SER A 51 -2 2 242 19.169 -5.150 5.42 OG SER A 51 -19 799 17.695 -4.429 22.03 CB SER A 51 -19 880 19.078 -4.315 10.76 CA SER A 51 - 2 1 326 19.519 -4.081 9.79 C SER A 51 - 2 1 223 21.032 -3.877 9.60 0 SER A 51 - 2 1 240 21.480 -2.721 12.84 N TYA 321 - 1 0 636 13.667 -4.206 16.47 OH TYA 321 -8 652 17.337 0.377 10.30 CD2 TYA 321 -9 066 16.694 -3.194 8.43 CE2 TYA 321 -8 450 16.999 -1.983 8.53 CZ TYA 321 -9 211 17.030 -0.825 10.25 CE1 TYA 321 - 1 0 581 16.747 -0.867 12.25 CD1 TYA 321 - 1 1 169 16.407 -2.086 11.85 CG TYA 321 - 1 0 426 16.387 -3.266 10.78 CB TYA 321 - 1 1 130 16.053 -4.553 1 1 .0 0 CA TYA 321 - 1 1 665 14.623 -4.652 15.20 C TYA 321 -1 2 056 14.339 - 6 .1 1 0 17.86 01 TYA 321 -13 099 14.928 -6.481 18.20 02 TYA 321 - 1 1 343 13.592 -6.810 17.88 0 HOH A 601 -17 519 15.016 -9.176 31.97 180 HG48 N GLY A 48 -19 910 18.305 -10.647 15.95 CA GLY A 48 -19 485 18.982 -9.439 17.66 C GLY A 48 -2 0 450 20.019 - 8 .8 8 8 20.28 0 GLY A 48 -2 0 108 20.618 -7.841 21.41 N TYA 321 -1 0 840 13.629 -3.984 22.65 OH TYA 321 -8 558 17.439 0.357 15.48 CD2 TYA 321 -8 962 16.823 -3.212 15.77 CE2 TYA 321 -8 377 17.175 -2.004 15.97 CZ TYA 321 -9 126 17.092 -0.835 16.95 CE1 TYA 321 -1 0 452 16.666 -0.879 17.95 CD1 TYA 321 -1 1 010 16.305 -2.105 18.40 CG TYA 321 -1 0 278 16.377 -3.290 16.82 CB TYA 321 -1 0 912 16.006 -4.605 18.39 CA TYA 321 -1 1 656 14.666 -4.634 20.68 C TYA 321 -1 1 990 14.272 -6.075 21.76 01 TYA 321 -13 020 14.813 -6.551 22.02 02 TYA 321 -1 1 260 13.471 -6.674 21.90 0 HOH A 604 -15 924 18.973 -11.884 24.88 181 Appendix m . Coordinate Shifts and Temperature Factor Differences This appendix consists of five sets of diagrams which aim to show changes in the overall separation of atoms and associated temperature factors for each mutant structure when compared with the wild—type TyrTS. Each set contains two plots of the r.m.s. separation in A between equivalent mainchain and side chain atoms. These are followed by two additional plots of the differences in average mainchain and side chain temperature factors (all four plots produced by the program DIFRES; T. Skarzynski, personal communication). A fifth diagram displays the difference distance matrix described below. These diagrams are repeated for each mutant. A final summary of these data is given in Table AIII.l located at the end of this appendix. The difference distance matrix aims to show changes in the relative distances of a —carbons between two similar structures (T. Skarzynski and A.J. Wonacott, submitted). The construction of this matrix is based upon the original difference maps first introduced by Phillips (1970) and Ooi and Nishikawa (1973). As stated by Janin and Chothia (1985), '[t]o draw such maps, the distances r(j between the a —carbon atoms of residues numbered i and j in the amino acid sequence are calculated and plotted against the two indices as a matrix which, for convenience, is usually represented as a two-dimensional contour map with a line joining points where rty has a given value say, loA.' The resulting patterns can be identified as particular secondary structure elements (i.e. a —helices, 0—pleated sheets) or domains. The 'difference* distance matrices, produced by the program 'Ooi—N' (T. Skarynski, personal communication), arise from calculation of the difference between equivalent rty's of two different structures. A continuous line is drawn when the difference, exceeds a particular value in angstroms. The difference matrices shown in this appendix applied the following criteria: = rjj wild—type — mutant ^ ± 0.2X. It is important to note that Ar^ may take on a positive or negative value strictly dependent upon the magnitude of the first vector (wild—type) relative to the second 182 (mutant). Hence, according to the relationship above an increase in the separation of two atoms in the mutant structure compared to the wild—type structure will appear as a negative value for Ar^y, shown in the upper left hand portion of these matrices. Likewise, a decrease in atomic separation appears as a positive value for Ar{*y, shown in the lower right hand portion of these matrices. Single points indicate movements associated with an isolated atom. Closed contours demonstrate movements of continuous chains such as those associated with residues 45 —55 in the TyrTS structure (shown, for example, in the upper left-hand portion of diagram AIH.4e) or entire domains as shown by the comparison of apo— and holoenzyme structures of glyceraldehyde phosphate dehydrogenase (T. Skarzynski and A.J. Wonacott, submitted). A m .l TP51 - WUd-type b. The r.m.s. separation between equivalent side chains. c. The difference in temperature factors between equivalent mainchain atoms. ll I..I llij I ,nl. Ii, j lllilti \ ll llil.i ll . i i Jl ll,, ,i 1 IlJIjI jIJIJLJi] ■ T n p F |T | It H ™ Till r IT l * y | T r i "i i i i r "I " 1 ’ 1 "T " 1 "1 V 1 ' 1 A | A50 A100 A150 A200 A252 A302 Residue number d. The difference in temperature factors between equivalent side chains. AIII.l (continued) 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 300 280 260 240 220 200 180 160 140 120 100 80 60 40 20 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 e. The TP51 — Wild—type difference distance matrix. AHI.2 TA51 - W ild-type Residue number a. The r.m.s. separation between equivalent mainchain atoms. b. The r.m.s. separation between equivalent side chains. . The difference in temperature factors between equivalent mainchain atoms. Residue number d. The difference in temperature factors between equivalent side chains. A m . 2 (continued) 80 100 120 140 160 180 200 220 240 260 280 300 300 280 260 240 220 200 180 160 140 120 100 80 60 40 20 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 e. The TA51 — Wild—type difference distance matrix. AIII.3 TS51 — Wild “ type b. The r.m.s. separation between equivalent side chains. c. The difference in temperature factors between equivalent mainchain atoms. d. The difference in temperature factors between equivalent side chains. AIII.3 (continued) 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 300 280 260 240 220 200 180 160 140 120 100 80 60 40 20 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 e. The TS51 — Wild—type difference distance matrix. 189 Ain.4 TG51 - Wild-type b. The r.m.s. separation between equivalent side chains. c. The difference in temperature factors between equivalent mainchain atoms. d. The difference in temperature factors between equivalent side chains. Am.4 (continued) 60 80 100 120 140 160 180 200 220 240 260 280 300 300 280 • < u * 260 240 220 200 180 160 140 120 100 80 60 40 20 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 e. The TG51 — Wild—type difference distance matrix. * Ain.5 HG48 - Wild-type 191 A150 A200 Residue number A302 a. The r.m.s separation between equivalent mainchain atoms. A 100 AI 50 A200 Residue number A 302 b. The r.m.s. separation between equivalent side chains c. The difference in temperature factors between equivalent mainchain atoms. d. The difference in temperature factors between equivalent side chains. Am.5 (continued) 80 100 120 140 160 180 200 220 210 260 280 300 300 280 260 240 220 200 180 160 140 120 100 80 60 40 20 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 e. The HG48 — Wild—type difference distance matrix. Table A in.l Summary of Structural Differences Wild-type TP51 TA51 TS51 TG51 HG48 Ha i rtcha i ns no. of atoms 1265 1265 1265 1268 1268 Overall r.m.s 0 . 1 0 0.18 0 . 1 2 0.18 0.14 0 separation (A) • o CO Mean x.y^z o, CM - -.03, -.07, -.03, o 00 o sh ifts (A) -. 0 1 , 0 -.03,.01 .03,.02 0 , .0 1 Overall diffs. 1.85 2 . 1 1 0.78 3.62 2.25 of temperature factors (A2) Mean value 14.6 16.42 16.68 15.35 18.18 16.81 of temperature factors (A2) Side ch a in s no. of atoms 1192 1192 1192 1192 1189 Overall r.m.s. 0.15 0.25 0.19 0.25 0 . 2 1 0 separation (A) Mean x,y^z, o, . 1 0 , -. 0 2 , -.06, -.03, sh ifts (A) 0 , 0 .08,.01 -.03,0 .04,.02 . 0 1 , . 0 1 Overall diffs. 0.60 0.75 -.014 2.27 0.53 of temperature factors (A2) Mean value 17.0 17.55 17.70 16.81 19.22 17.48 of temperature factors (A2) A ll atom s no. of atoms 2457 2457 2457 2460 2457 Overall r.m.s. 0.13 0 . 2 2 0.16 0 . 2 2 0.17 0 separation (A) Mean x.y^z, o, .09, -. 0 2 , -.06, -.03, sh ifts (A) 0 , 0 .08,.02 -.03,.01 .03,.02 0 , .0 1 Overall diffs. 1.24 1.45 0.34 2.96 1.42 of temperature factors (A2) Mean value 15.8 16.99 17.19 16.09 18.68 17.14 of temperature factors (A2) 194 REFERENCES Ahern, T.J., Casal, J.I., Petsko, G.A. and Klibanov, A.M. 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