molecular determinants of nonaqueous biocatalysis

DianaAndreiaPereiraLousa

DissertationpresentedtoobtainthePh.DdegreeinBiochemistryInstitutodeTecnologiaQumicaeBiolgica|UniversidadeNovadeLisboa

Oeiras,March,2013

MoleculardeterminantsofnonaqueousbiocatalysisAcomputationalanalysis

DianaAndreiaPereiraLousa

DissertationpresentedtoobtainthePh.DdegreeinBiochemistryInstitutodeTecnologiaQumicaeBiolgica|UniversidadeNovadeLisboa

Oeiras,March,2013

MoleculardeterminantsofnonaqueousbiocatalysisAcomputationalanalysis

Molecular determinants of

nonaqueous biocatalysis

A computational analysis

Diana Andreia Pereira Lousa

Supervisors: Professor Cludio M. Soares and Doctor Antnio M. Baptista

Dissertation presented to obtain the Ph.D degree in Biochemistry

The work presented in this thesis was financed by Fundao para a Cincia e a

Tecnologia through grant SFRH/BD/28269/2006, with the support from the

European Social Fund.

Contents

5

Contents

Acknowledgments 9

List of publications 11

Papers presented in this thesis 11

Abstract 13

Resumo 17

List of symbols and abbreviations 23

Abbreviations 23

Latin symbols 24

Greek symbols 24

1 Introduction 27

1.1 Biomolecular catalysis: How do enzymes work? 28

1.1.1 Historical perspective 28

1.1.2 Current perspective(s) 30

1.2 Enzymatic catalysis in nonaqueous media 35

1.2.1 Structural and dynamical properties of enzymes in nonaqueous solvents 38

1.2.2 Enzyme activity and selectivity in nonaqueous solvents 39

1.2.3 The role of counterions 42

1.2.4 pH effects 45

1.2.5 Ligand imprinting 46

1.3 Simulation studies of enzymes in nonaqueous solvents 47

1.3.1 Setup challenges 49

1.3.2 Protein structure 50

1.3.3 Protein flexibility 52

1.3.4 Formation of salt bridges and intra-protein hydrogen bonds 53

1.3.5 Protein-solvent interactions 54

1.3.6 Effect of water concentration and solvent polarity 56

1.3.7 The role of counterions 60

1.3.8 Enzyme activity and enantioselectivity 61

1.3.9 Lipase interfacial activation 63

1.3.10 Simulation studies of enzymes in ionic liquids 64

1.3.11 Simulation studies of enzymes in supercritical fluids 66

1.4 Scope of the present thesis 68

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2 Theory and methods 71

2.1 Biomolecular modelling and simulation 72

2.2 Molecular mechanics 74

2.2.1 Molecular mechanics force fields 74

2.2.2 Bonded interactions 76

2.2.3 Nonbonded interactions 77

2.3 Energy minimization 78

2.4 Molecular dynamics simulations 80

2.4.1 Integration algorithms 83

2.4.2 MD simulations with periodic boundary conditions 84

2.4.3 MD simulations at constant temperature and/or pressure 85

2.4.4 Free energy calculations using MD simulations 87

2.5 Molecular docking 89

2.5.1 Docking algorithms 90

2.5.2 Scoring functions 91

2.4 Prediction of protonation states using continuum electrostatics and Monte Carlo simulations 93

3 Interaction of counterions with subtilisin in acetonitrile: Insights from molecular dynamics simulations 99

3.1 Abstract 101

3.2 Introduction 101

3.3 Materials and methods 104

3.3.1 Calculation of the potentials of mean force (PMFs) 104

3.3.2 MD simulations 105

3.3.3 Protein structures used in the MD simulations 105

3.3.4 Modeling protein protonation equilibrium 105

3.3.5 Setup for MD simulations 107

3.4 Results and discussion 108

3.4.1 Potentials of mean force between the cations, Cs+ and Na+, and the anion, Cl, in solvents with different polarities 108

3.4.2 Determination of the protonation state of ionisable residues at pH 6.5 109

3.4.3 Stability of the simulations 109

3.4.4 Comparison of Xray and docking ion binding sites 111

3.4.5 Occupancy of the ion binding sites during MD simulations 113

3.4.6 Distribution of counterions on the enzyme surface in acetonitrile simulations 116

3.4.7 Distribution of counterions on the enzyme surface in water simulations 122

3.4.8 Analyzing the effect of different cations on the activity of subtilisin 125

3.5 Conclusions 128

Contents

7

4 Analyzing the molecular basis of enzyme stability in ethanol/water mixtures using molecular dynamics simulations 131

4.1 Abstract 133

4.2 Introduction 134

4.3 Materials and methods 136

4.4 Results and discussion 138

4.4.1 Structural stability of the proteins in water and ethanol/water simulations 138

4.4.2 Protein-ethanol interaction 145

4.4.3 Comparing the behavior of wild type and C58G mutant of pseudolysin 149

4.5 Conclusion 150

4.6 Acknowledgements 152

5 Structural determinants of ligand imprinting: A molecular dynamics simulation study of subtilisin in aqueous and apolar solvents 153

5.1 Abstract 155

5.2 Introduction 156

5.3 Materials and methods 158

5.3.1 Protein structure selection 158

5.3.2 Determination of protonation states 158

5.3.3 Docking of the inhibitor 159

5.3.4 Molecular dynamics simulations 160

5.3.5 Hydration conditions in hexane simulations 161

5.3.6 Selection of counterion positions 162

5.4 Results and discussion 162

5.4.1 Docking of the inhibitor 164

5.4.2 Stability of the simulations 165

5.4.3 Effect of pretreating the enzyme with the ligand: hexane vs. water simulations 165

5.4.4 Why does ligand imprinting occur in hexane but not in water? 169

5.5 Conclusion 171

5.6 Acknowledgements 172

6 Final discussion 173

6.1 Protein-ion interactions in nonaqueous solvents 174

6.2 Protein stability in ethanol/water mixtures 178

6.3 Ligand imprinting 179

Appendix A: Supporting information for chapter 3 183

A.1.1 Protocol for selecting counterion positions using molecular docking 183

A.1.2 Methodology used to randomly distribute Cs+ and Cl- ions in the simulations performed in water with 1.5 M of salt 186

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A.1.3 Protocol for modeling protein protonation equilibrium 187

A.2. Results and discussion 190

A.2.1 Potentials of mean force between the cations, Cs+ and Na+, and the anion, Cl-, in solvents with different polarities 190

A.2.2 Determination of protonation of ionizable residues at pH 6.5 192

A.2.3 Evolution of the protein structure in acetonitrile and water simulations 194

A.2.4 Electrostatic surface maps of subtilisin in the crystal environment and in solution 196

A.2.5 Radial distribution function of Cl- around the N2 of H64 197

A.3 Movies 198

Appendix B: Supporting information for chapter 4 199

B.1. Methods 199

B.1.2 System preparation for MD simulations 199

B.1.3 Methodology used in the determination of protonation states 201

B.2 Results 202

B.2.1 Analysis of rigid body motions between the domains of the proteins under study 202

B.2.2 Contact area between water molecules and the protein 204

B.2.3 Distribution of the water molecules around the protein 205

B.2.4 Distributions of the alcohol and alkyl moieties of the ethanol molecule around the protein 206

B.2.5 Comparison of the thermolysin residues that interact most frequently with ethanol in our simulations with the binding sites of isopropanol determined in a previous X-ray study 207

B.2.6 Areas of the histogram peaks 208

B.2.7 Comparing the behavior of wild type and C58G mutant of pseudolysin 209

Appendix C: Supplementary information for chapter 5 211

C.1 Methods 211

C.1.1 Protocol for selecting counterion positions 211

C.2 Results 214

C2.1 Protein stability 214

C.2.2. Behavior of the loops surrounding the S1 pocket 218

C.3 Movies 222

Bibliography 223

Acknowledgments

9

Acknowledgments

First, I would like to thank my supervisors, Prof. Cludio M. Soares and Dr.

Antnio M. Baptista, for teaching me everything I know about science, and for

their support and friendship. I have to thank them for making me believe,

even when I couldnt see the light in the end of the tunnel. I am convinced

that this is one of the most important qualities of a supervisor.

I am grateful to my colleagues from the Protein Modeling and Molecular

Simulation groups for all their help and friendship, and for making this fun. I

am proud to be part of the most eccentric (a.k.a. nerd) group of ITQB.

I also want to thank my parents, who always supported my decisions. I always

felt that I could choose to be whatever I wanted (although most of the time I

didnt know what that was).

I am thankful to my brothers for making me realize, early in life, that my

athletic skills were so bad that I could only become an intellectual. By the

time I was eight, after three consecutive last places in running events, I was

pretty sure sports werent my future. My brothers, of course, made sure I

would stay on the right path, by constantly reminding me of my impressive

record of three last places in a row.

I want to thank my friends and family for being there for me when I needed

them.

Finally, I acknowledge Instituto de Tecnologia Qumica e Biolgica for the

excellent working conditions and Fundao para a Cincia e a Tecnologia for

funding through grant SFRH/BD/28269/2006.

List of publications

11

List of publications

Papers presented in this th