Luận án tiến sĩ: Computational studies to understand molecular regulation of the TRPC6 calcium channel, the mechanism of purine biosynthesis, and the folding of azobenzene oligomers

273 8.14 Contour plots of the free energy at 300 K projected onto the dihedral angle 1 and dihedral angle 2 reaction coordinates for REMD simulation of 7.2.. 276 8.16 Contour plots of th

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COMPUTATIONAL STUDIES TO UNDERSTAND MOLECULAR REGULATION OF THE TRPC6 CALCIUM CHANNEL,

THE MECHANISM OF PURINE BIOSYNTHESIS,

AND THE FOLDING OF AZOBENZENE OLIGOMERS

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Professor Russell M Pitzer

_

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UMI Number: 3241692

3241692 2007

UMI Microform Copyright

ProQuest Information and Learning Company

300 North Zeeb Road P.O Box 1346 Ann Arbor, MI 48106-1346

by ProQuest Information and Learning Company

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ABSTRACT

Different computational chemistry methods were applied to study a variety of problems at the molecular level These problems concern protein-protein interactions, the mechanism of reaction for enzymatic purine biosynthesis, structural interconversion

in non-natural oligomeric folding, and carbohydrate synthesis

Transient receptor potential-canonical 6 (TRPC6) calcium channels are currently the subject of intense investigation for their role in modulating smooth muscle tone in blood vessels and lung tissue Binding of a protein, FKBP12, is a prerequisite for the formation of a multiprotein complex involved in channel regulation To study the elements of molecular recognition in FKBP12 for binding to the TRPC6 intracellular domain, 20 nanosecond molecular dynamic simulations were performed on the complex

of FKBP12 and a peptide model of the wild-type TRPC6 intracellular domain, a

phosphorylated Ser768 analog of the wild-type peptide as well as Ser768Asp and

Ser768Glu mutants The phosphorylated peptide demonstrated the greatest binding affinity by the MM-GB/SA method, due to the strong interaction between the phosphate group and two lysine (Lys44 and Lys47) residues of FKBP12 at the binding site These trajectories also revealed transient, non-simultaneous interactions with the ε-NH3⊕ group

of these lysine residues This feature was not observed in simulations containing the

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identified important additional structural elements necessary for this protein-protein interaction

Potential catalytic reaction mechanisms of the enzyme PurE Class I, which catalyzes the transformation from N5-carboxyaminoimidazole ribonucleotide (N5-CAIR)

to 4-carboxyaminoimidazole ribonucleotide (CAIR) in the purine biosynthetic pathway, were investigated by density functional theory (DFT) methods The potential energy surfaces (PES) of model processes for these enzymatic reactions have been explored, and have aided in identifying the most energetically feasible pathway Calculations using a simplified model system, containing only the essential atoms involved in the chemical process, revealed seven potential reaction pathways for transformation of N5-CAIR to CAIR The experimental results exclude four of these pathways Two of the remaining three pathways involve deprotonation of one carbon atom (C4) of the imidazole ring This process makes the relative energy of transition states of these two pathways higher than the third pathway

The full N5-CAIR structure was studied via PES calculations, including

consideration of the ribose-5-phosphate unit and its different charge states There are 48 structures of N5-CAIR and CAIR regarding the different protonation states of the

substrate Four reaction pathways were identified based on the available structures after optimization One pathway involved a cationic substrate Two involved anionic

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substrates A fourth one involved a neutral substrate These four pathways have similar PES to their counterparts in the simplified model

The cationic pathway is the most favorable pathway in both the simplified and full reaction models In this pathway, an intramolecular proton donor/acceptor is

required before and after migration of the carboxyl group (-CO2H) According to the spatial arrangement of catalytic amino acids at the active site of the PurE Class I crystal structure (PDB ID: 1D7A), a conserved histidine 45 (His45) residue could be such a proton donor/acceptor Based on these results, a stepwise enzymatic reaction mechanism for PurE Class I is proposed First, His45 at the PurE Class I active site protonates the amide nitrogen of N5-CAIR Second, the carboxyl group migrates to carbon 4 (C4) with concomitant C-C bond formation This step generates the protonated CAIR intermediate

In the final step, His45 deprotonates the protonated CAIR intermediate to produce the final product, CAIR, and regenerates its initial state For the first time, an atomistic description of the PurE Class I enzyme catalytic mechanism is provided This

information can be applied to the development of transition state analogs and based inhibitors of PurE Class I These newly designed molecules could be used as potential new drug leads for optimization by synthetic and medicinal chemistry

mechanism-Foldamers are defined as unnatural polymers/oligomers with a well-defined, compact, three-dimensional folding capability Azobenzene units are common linkages

in foldamer designs Four alternating pyridinedicarboxamide/m-(phenylazo)azobenzene

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oligomers which could fold into both right- and left-handed helices were studied

computationally for their dynamical properties Two helices were shown as the global minimum among the conformations generated by Monte Carlo simulation Extended conformations have higher potential energies than compact ones Molecular dynamics simulations (100 ns) at a single temperature were also performed to study the

interconversion process between two helices of these oligomers However, the molecules were trapped at the local minimum, and no interconversion was observed To overcome this difficulty, replica-exchange molecular dynamic (REMD) simulations which apply a parallel tempering algorithm were performed on the azobenzene oligomers Both right- and left-handed helices were successfully sampled in the simulation for all four

oligomers Careful investigation of REMD trajectories revealed twisted conformations as intermediate structures in the interconversion pathway between two helices

The temperature weighted histogram analysis method (T-WHAM) was applied on the REMD simulation results to generate contour maps of the potential of mean force (PMF) Atomic pair distances and dihedral angles were used as reaction coordinates for PMF contour plots Analysis showed that right- and left-handed helices are equally sampled in REMD simulations In large oligomers, both right- and left-handed helices could be adopted by different parts of the molecule simultaneously The interconversion between two helices could occur in the middle of the helical structure, which is not necessarily at the end of the molecule

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Oligosaccharides play a variety of important roles in a number of biological events, and the stereoselective synthesis of carbohydrates is critical Computational studies have been done to understand the mechanism of the regioselective epoxide ring-opening process catalyzed by the presence of (-)-sparteine in the synthesis of β-

arabinofuranosides Using a simple diamine, N,N,N’,N’-tetramethylpropanediamine, to

mimic (-)-sparteine, DFT was applied to search for transition states connected to two unique epoxide ring-opening events for attack at two different carbons of the epoxide ring However, the reaction barriers calculated by DFT in this simplified model could not explain the observed experimental regioselectivity DFT single-point energy calculations using (-)-sparteine lead to similar results to those of the simplified model Molecular dynamics (MD) methods were applied to simulate five systems containing the (-)-

sparteine-Li+ complex and different substrates MD simulations revealed interesting conformational changes of the (-)-sparteine unit By switching between two conformers, (-)-sparteine could push Li+ toward the epoxide oxygen in a catalytically useful fashion Since the relative orientation of (-)-sparteine with respect to the furanose ring is largely influenced by steric effects due to the substituent group at the C5 position of substrate, (-)-sparteine helps Li+ selectively approach the epoxide oxygen on one face, which leads

to specific ring-opening at the C3 position In addition to traditional MD, ab initio

molecular dynamics methods were applied to simulate four systems for the epoxide opening process The conformations of (-)-sparteine during the ring-opening process

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ring-agree with the observation in our earlier classical MD simulations, and this conformational control provides the origin of the experimental selectivity

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Dedicated to my beautiful wife Jin

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ACKNOWLEDGMENTS

In the past five years, my advisor Dr Christopher M Hadad gave me tremendous support for my study and research at the Ohio State University Without his help, none of this would have been possible His enthusiasm about science and high productivity in research set up a lifetime model for me to follow

I also would like to thank Dr Russell M Pitzer for allowing me to attend his group meetings in the past year This enjoyable experience gave me lots of education in quantum chemistry Many other faculty members in the physical division also educated

me about physical chemistry I would give my special thanks to Dr James V Coe as my committee member

Dr Hadad’s research group members are always friends and colleagues for me

Dr John C Hackett provided much help in my research and in critical reading of my manuscripts Ms Carrigan J Hayes and Mr Matthew P DeMatteo also helped me with their collaboration and critical reading I also want to thank all other group members for their friendship and help

Financial support from the National Science Foundation and the National Institute of Health is gratefully acknowledged

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My mother, Mrs Xiuying Liang, my father, Mr Junhe Tao, my mother-in-law, Mrs Qiaoyun Liu, and my father-in-law, Mr Binyan Liu all gave me strong support when I was far away from my home country, People’s Republic of China

I want to dedicate this dissertation to the most important person in my whole life,

my beautiful wife Dr Jin Liu Your support was always with me during the completion

of this dissertation Your intelligence sheds lights on many of my scientific problems Your enthusiasm about life always encourages me to move forward Any achievement would be meaningless for me without your sharing You make me the luckiest person in the whole world with your endless love, support, and encouragement No words are adequate enough to express my gratitude A journey with you is the most beautiful thing

I can ask for in my life

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Peking University, Beijing, China

2001 – 2005 Graduate Teaching and Research Associate,

The Ohio State University

PUBLICATIONS

Research Publications

1 Mendlik, M T.; Tao, P.; Hadad, C M.; Coleman, R S.; Lowary, T L “Synthesis

of L-Daunosamine and L-Ristosamine Glycosides via Photoinduced Aziridination

Conversion to Thioglycosides for Use in Glycosylation Reactions” J Org Chem 2006,

71, 8059-8070

2 Tao, P.; Lai, L “Protein ligand docking based on empirical method for binding

affinity estimation.” J Comput.-Aided Mol Des 2001, 15, 429-46

3 Tao, P.; Wang, R.; Lai, L “Calculating partition coefficients of peptides by the

addition method.” J Mol Mod 1999, 5, 189-195

4 Tao, P.; Wang, R.; Lai, L “Calculation of peptide's partition coefficients by

amino acid addition model.” Wuli Huaxue Xuebao 1999, 15, 449-453

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FIELDS OF STUDY Major Field: Chemistry

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TABLE OF CONTENTS

P a g e

Abstract ii

Dedication viii

Acknowledgments ix

Vita xi

List of Tables xviii

List of Figures xxiii

Chapters: 1 Introduction 1

1.1 TRPC ion channels 2

1.2 N5-CAIR mutase mechanism in purine biosynthesis pathway 4

1.3 Foldamers 8

1.4 Regioselective ring-opening of epoxides in 2,3-anhydrosugars 12

1.5 References for Chapter 1 14

2 Computational techniques 21

2.1 Density functional theory 21

2.2 Monte Carlo simulations 24

2.3 Molecular dynamics simulations 26

2.4 Quantum molecular dynamics 28

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3 Molecular determinants of TRPC6 channel recognition by FKBP12 33

3.1 Introduction 33

3.2 Computational methods 37

3.2.1 TRPC6 peptide docking 37

3.2.2 Molecular dynamics simulation of FKBP12-TRPC6 peptide complexes and separated FKBP12 and TRPC6 peptides 39

3.2.3 MM/GB-SA free energy of binding calculations 40

3.3 Results and discussion 41

3.3.1 TRPC6 peptide docking 41

3.3.2 RMSD fluctuation of complexes 45

3.3.3 Residue fluctuation in each simulation 47

3.3.4 Binding free energy calculations by MM-GBSA based on Single-Trajectory 54

3.3.5 Free energy of binding calculations of FKBP12 with unphosphorylated and phosphorylated wild-type peptide complexes based on Single-Trajectory 59

3.3.6 Free energy of binding calculations of Ser768Asp and Ser768Glu mutants based on Single-Trajectory 60

3.3.7 Entropic contribution of each complex based on Single-Trajectory 61

3.3.8 Free energy of binding calculations of WT complex based on Three-Trajectory 63

3.3.9 Free energy of binding calculations of mutant complexes based on Three-Trajectory 64

3.3.10 Decomposition analysis of the free energy of binding based on Single-Trajectory 65

3.3.11 Decomposition analysis of the free energy of binding based on Three-Trajectory 73

3.3.12 Spatial distribution of residues with major contributions to the free energy of binding 73

3.3.13 Interactions among Lys44, Lys47 and Residue768 75 3.3.14 Comparison of RMSD fluctuation between bound and unbound peptides 81

3.3.15 Comparison of RMSD fluctuation between bound and unbound FKBP12 85

3.4 Conclusions 87

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4 Computational studies of the N5-CAIR mutase mechanism in the purine

biosynthesis pathway 97

4.1 Introduction 97

4.2 Computational Methods 101

4.3 Results and Discussions 103

4.3.1 Simplified Models 103

4.3.2 Thermochemistry for transformation of AMI to AMIC 103

4.3.3 From MICA to AMIC through cationic or anionic pathways: a brief glance 106

4.3.4 Exploring the PES of carboxyl unit migration 111

4.3.5 Complete mechanism involved with enzyme active site starting with MICAp1 122

4.3.6 Verification of the Simplified Model 128

4.4 Conclusions 131

5 Potential energy surface of N5-CAIR mutase catalyzed reaction 138

5.1 Introduction 138

5.3 Results 140

5.3.1 Model structures 140

5.3.2 Optimization in the gas phase 142

5.3.3 Aqueous phase calculation with the PCM method 148

5.3.4 PCM calculations in chlorobenzene 150

5.3.5 Transition states search related to previous calculations 153

5.4 Discussion 161

5.4.1 Thermochemistry of N5-CAIR and CAIR 161

5.4.2 Transition States of Carboxyl Group Migrations 164

5.5 Conclusions 167

6 Substituent effects in the N5-CAIR mutase mechanism 170

6.1 Introduction 170

6.3 Results 172

6.3.1 Analogs with either R1 or R2 as a methyl group 174

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6.3.2 Analogs with either R1 or R2 as a trifluoromethyl group 180

6.3.3 Analogs with either R1 or R2 as a cyano group 186

6.3.4 Analogs with either R1 or R2 as a carboxylic ester group 192

6.3.5 Analogs with either R1 or R2 as a nitro group 199

6.4 Conclusions 206

7 Folding of helical structures of alternating pyridinedicarboxamide/M-(phenylazo) azobeneze oligomers 210

7.2 Computational methods 213

7.3 Results and Discussion 216

7.3.1 Monte Carlo Conformational Search of 7.1 216

7.3.2 Monte Carlo Conformational Search of 7.2 226

7.3.3 Molecular Dynamics of 7.1 232

7.3.4 Molecular Dynamics of 7.2 237

7.3.5 Replica Exchange Molecular Dynamics Simulation (REMD) of 7.1 241

7.3.6 Replica Exchange Molecular Dynamics Simulation (REMD) of 7.2 245

7.4 Conclusions 247

8 Potential of mean force of oligomer folding 253

8.2 Methods 254

8.3 Results and Discussion 257

8.3.1 Replica Exchange Diagnostics 257

8.3.2 REMD simulations of 7.1 263

8.4 Conclusions 298

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9 Computational investigations of the regioselective ring-opening of

epoxides in 2,3-anhydrosugars: the role of (-)-sparteine 301

9.1 Introduction 301

9.2 Methods 305

9.2.1 Epoxide ring-opening reactions 305

9.2.2 Molecular Dynamics simulation of sparteine, sugar ring and lithium system 306

9.2.3 CPMD simulation method 307

9.3 Results and Discussion 308

9.3.1 Gas-phase and PCM potential energy surfaces 308

9.3.2 Molecular Dynamics simulations 312

9.3.2.1 Molecular Dynamics Simulation for MD9.6 (C5=O-) 313

9.3.2.2 Molecular Dynamics Simulation for MD9.7 (C5=OH) 317

9.3.2.3 Molecular Dynamics Simulation for MD9.8 (C5=NH2) 319

9.3.2.4 Molecular Dynamics Simulation for the System MD9.9 (C5=C6H5CH2OCH2O-) 321

9.3.2.5 Molecular Dynamics Simulation for the System MD9.10 (C5=C 5 NH 4 CH 2 O-) 325

9.3.3 ab initio Molecular Dynamics Simulation Results 326

9.4 Conclusions 332

Bibliography 338

Appendix A Supporting information for Chapter 3 361

Appendix B Supporting information for Chapter 4 367

Appendix C Supporting information for Chapter 5 388

Appendix D Supporting information for Chapter 7 403

Appendix E Supporting information for Chapter 9 410

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LIST OF TABLES

3.1 Energy scores (kcal/mol) from DOCK for the preferred modes of TGF-β

receptor Type I and TRPC6 peptides binding to the FKBP12 receptor 44

3.2 Binding free energy components of FKBP12 and wild peptides

complexes 583.3 Calculated entropic change (Delta) by NMODE 62

4.1 Thermochemistry (kcal/mol) of model reactions for AMI to MICA and

AMIC 104

4.2 Energy comparison of different products generated via methylation,

carboxylation and protonation reactions of AMI (B3LYP/6-31+G(d)) 109

4.3 Transition-state calculation results for different protonation states of

MICA 1165.1 DFT Computational results in the gas phase 144

5.2 DFT single-point energetic results using the PCM model for water

(ε=78.39) at the B3LYP/6-31+G(d) level of theory (298 K, kcal/mol)

using the B3LYP/6-31+G(d) optimized geometry from the gas phase 149

5.3 DFT single-point energetic results using the PCM model for

chlorobenzene (ε=5.62) at the B3LYP/6-31+G(d) level of theory (298 K,

kcal/mol) using the B3LYP/6-31+G(d) optimized geometry from the gas

phase 1525.4 Transition states for carboxylate group migration using the full model 154

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6.2 Substituent calculation with R1=H, R2=CH3 178

6.3 Substituent calculation with R1=CF3, R2=H 181

6.4 Substituent calculation with R1=H, R2=CF3 184

6.5 Substituent calculation with R1=CN, R2=H 187

6.6 Substituent calculation with R1= H, R2 = CN 190

6.7 Substituent calculation with R1 = CO2CH3, R2 = H 193

6.8 Substituent calculation with R1 = H, R2 = CO2CH3 197

6.9 Substituent calculation with R1 = NO2, R2 = H 201

6.10 Substituent calculation with R1 = H, R2 = NO2 203

7.1 First 20 Conformers in Monte Carlo Simulations of 7.1 220

9.1 Transition-state Preferences for C3 vs C2 ring-opening of the epoxide for the anion, Li+, and tetramethylpropanediamine model at the B3LYP/6-311+G**//B3LYP/6-31G* level of theory (kcal/mol) 309

9.2 Transition-state Preferences for C3 vs C2 ring-opening of the epoxide for the anion, Li+, and (-)-Sparteine model at the B3LYP/6-311+G**//B3LYP/6-31G* level of theory (kcal/mol) 311

9.3 MD simulation details 312

A.1 MD simulation details 366

B.1 Energies for model reactions B3LYP/6-311+G(3df,2p)//B3LYP/6-31+G(d) 368

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B.2 Energies for methyl group addition products on different sites of AMI at

B3LYP/6-31+G(d) level of theory 369

B.3 Energies for carboxylate group addition products on different sites of AMI at

B.4 Energies for protonation products on different sites of AMI at

MICAp8 376

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B.14 CBS-QB3 Energies for structures along carboxylate group migration

through MICAp1 376

B.15 Energies for structures along deprotonation pathway of MICAp1

carboxylate migration product by histidine at B3LYP/6-31+G(d) level of

theory 377

B.16 Energies for structures along protonation pathway of MICAp1 by

histidine at B3LYP/6-31+G(d) level of theory 377

B.17 Energies for structures along carboxyl group migration pathway with the

complete N5-CAIR structure, and including the ribose-5-phosphate unit

at B3LYP/6-31+G(d) level of theory 378

C.1 Energies for N5-CAIR structures with code 000-(0,1, and 2) at

31+G(d) level of theory 392

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C.9 Energies for CAIR structures with code 000-(0,1, and 2) at

C.17 Energies for structures along the carboxylate migration pathway through

N5-CAIR 011-1 and CIAR 011-1 at B3LYP/6-31+G(d) level of theory 397

N5-CAIR 101-1 and CIAR 101-1 at B3LYP/6-31+G(d) level of theory 398C.20 Energies for structures along the carboxylate migration pathway through

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LIST OF FIGURES

1.1 Structure of TRPC6 channel and FKBP12 binding site 41.2 Structures of Purine, Adenine and Guanine 51.3 Biosynthetic sources of purine ring atoms 61.4 Structure of tetraazobenzene oligomer and unit cell of crystal structure 11

1.5 Synthesis of β-arabinofuranosides (1.4) from 2,3-anhydrosugar

glycosylating agents 1.1 and 1.2 13

3.1 Comparison of docked TFGβ receptor peptide (green) and crystal

structure (purple) 42

3.2 Comparison between structures of FKBP12~TFGβ receptor (Left),

FKBP12~docked TRPC6 peptide (middle) and FKBP12~docked

phosphorylated TRPC6 peptide (right) 43

3.3 RMSD of four complexes during 20ns MD simulations: complex with

wild type TRPC6 peptide (black), complex with phosphorylated wild

type TRPC6 peptide (red), complex with mutant Ser768Asp (green),

complex with mutant Ser768Glu (blue) 46

3.4 Average residue fluctuation of FKBP12 in MD simulations (analysis

based on 20ns simulation) 483.5 Fluctuation of amino acid residues in FKBP12- TRPC6 peptide

complexes 50

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3.6 Average residue fluctuation of each TRPC6 peptide in MD simulations

of four complexes (analysis based on 20ns) 55

3.7 Decomposition of binding free energies into each single residue from

3.12 Switching between Lys44 and Lys47 for forming salt bridge with

phosphate group from pSer768 783.13 Unbound and bound wild type peptide RMSD in each complex 823.14 RMSD of FKBP12 in four complexes and unbound mode 864.1 Reaction catalyzed by PurK and PurE Class I in prokaryotes 984.2 Reaction catalyzed by PurE Class II in eukaryotes 99

4.3 AMI, MICA and AMIC models represent AIR, N5-CAIR and CAIR,

respectively, where the CH3 group replaces the ribose-5-phosphate unit 1004.4 Model structures related to the cationic pathways for carboxyl group

migration 107

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4.5 Relative energies (kcal/mol) of N6 and C4 deprotonation products 1114.6 Possible protonation states of MICA for carboxyl group migration 1124.7 Different protonation states of histidine side chain 113

4.8 Potential energy surface of CO2 migration from amide nitrogen to C4 on

imidazole ring 115

4.9 The geometries of reactant, product and transition state of carboxyl

migration through MICA p1 1224.10 Proposed mechanism of PurE Class I (N5-CAIR Mutase) 1244.11 Active site of PurE Class I (PDB ID: 1D7A) 1254.12 Relative energies of His45 as proton acceptor/donor 127

4.13 Transition state for carboxyl group migration with full N5-CAIR

structure 128

4.14 Carboxyl group migration pathway with the complete N5-CAIR

structure, and including the ribose-5-phosphate unit 1305.1 Reaction catalyzed by PurE Class I 1395.2 N5-CAIR and CAIR Structures in Different Protonation States 141

5.3 CO2H Migration through N5-CAIR011-1 as calculated at the

B3LYP/6-31+G(d) level of theory in the gas phase 156

5.4 CO2H migration through N5-CAIR011-2 as calculated at the

B3LYP/6-31+G(d) level of theory in the gas phase 1575.5 CO2H migration through N5-CAIR101-1 as calculated at the B3LYP/6-

31+G(d) level of theory in the gas phase 159

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5.6 CO2H migration through N5-CAIR111-2 as calculated at the

B3LYP/6-31+G(d) level of theory in the gas phase 1605.7 Favorable reaction mechanisms of PurE Class I 1666.1 Analog structures of basic model for carboxylic group migration 173

7.1 Alternating Pyridinedicarboxamide/m-(Phenylazo)azobenzene

Oligomers 7.1 and 7.2 2117.2 Potential Energies of Monte Carlo Searched Conformers for 7.1 217

7.3 Left-handed helical structure with global minimum potential energy

surface for 7.1 2187.4 Crystal structure of 7.1 219

7.5 Overlap of 13 left-handed helices conformers of 7.1 among top 20 MC

structures 221

7.6 Overlap of seven right-handed helices conformers of 7.1 among top 20

MC structures 2227.7 Conformer 5808 (left) and 6289 (right) of 7.1 from MC results 2237.8 Distributions of distance between two carbons at either end of 7.1 2257.9 Potential Energies of Monte Carlo Searched Conformers for 7.2 227

7.10 Structure (half left-handed and half right-handed helical structures) with

global minimum potential energy surface for 7.2 2287.11 Four conformers of 7.2 with partially extended geometries 2307.12 Distributions of distance between two carbons at either end of 7.2 231

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7.13 RMSD of 100 ns MD simulation of 7.1 2337.14 Dihedral angle on one end of 7.1 2347.15 The distribution of distance between two carbons at one end of 7.1 2367.16 RMSD of 100 ns MD simulation of 7.2 2387.17 Four typical conformers of 7.2 from 100 ns MD Simulations 2387.18 Unfolding path of right-handed helix of 7.1 2407.19 Distribution of dihedral angles in replica 4 of 7.1 (T=295.9K) 2427.20 Mechanism of interconversion between two helices for 7.1 2447.21 Sampled conformers of 7.2 from REMD simulation 2468.1 Azobenzene oligomers with even numbers of pyridine rings 254

8.2 Temperature evolution of replica 6 in the REMD simulation of Oligomer

7.1 2598.3 Potential energy distribution of replica 6 in the REMD simulation of 7.1 260

8.4 Potential energy histograms of 12 replicas in the REMD simulation of

7.1 2618.5 Exchange rate among replicas in the REMD simulation of 7.1 2628.6 Definitions of dihedral angles and distances of 7.1 for WHAM analysis 2638.7 Contour plots of the Gibbs free energy (G) at 300 K projected onto the

dihedral angle 1 and dihedral angle 2 reaction coordinates in 7.1 265

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8.8 Representative conformers with M and P configurations of 7.1 266

8.9 The contour plots of the free energy at 300 K projected onto the distance

1 and distance 2 reaction coordinates for 7.1 268

8.10 Eigenvalues from the accumulated principal component analysis (PCA)

for 7.1 270

8.11 Contour plots of the free energy at 300 K projected onto PCA state 1 and

3 for 7.1 2718.12 Major movements represented by PCA state 1 and 3 of 7.1 2728.13 Definitions of dihedral angles and distances of 6.2 for WHAM analysis 273

8.14 Contour plots of the free energy at 300 K projected onto the dihedral

angle 1 and dihedral angle 2 reaction coordinates for REMD simulation

of 7.2 274

8.15 Conformer of 7.2 located at the off-diagonal attraction basin on the right

hand side in Figure 8.14 276

of 7.2 278

of 7.2 2798.18 Two Configurations of 7.2 2808.19 Contour plots of the free energy at 300 K projected onto the distance 1

and distance 2 reaction coordinates for REMD simulation of 7.2 282

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of 8.1 285

of 8.1 287

8.23 Definitions of dihedral angles and distances of 8.2 for WHAM analysis

Eight dihedral angles are defined by carbon atoms from pyridine rings

and ring centers of azobenzenes 288

of 8.2 290

of 8.2 291

of 8.2 293

of 8.2 295

8.28 Contour plots of the free energy at 300 K projected onto the distance 1

and distance 2 reaction coordinates for REMD simulation of 8.2 2979.1 Furanose Rings with Different C5 Substituents 3079.2 Transition states with N,N,N’,N’-tetramethylpropanediamine for C2 and

C3 attack of the ring-opening of the epoxide at the B3LYP/6-31G* level 309

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9.3 Transition states with (-)-sparteine for C2 and C3 attack of the

ring-opening of the epoxide at the B3LYP/6-31G* level 3119.4 Atomic pair distance fluctuation in MD simulation of MD9.6 314

9.5 N1-Li-N2 angle for (-)-sparteine and Li+ complexes for (a) MD9.6

(black) and MD9.7 (red); (b) MD9.8 (black), MD9.9 (red) and MD9.10

(green) 314

9.6 Distribution of exocyclic dihedral angles from MD simulations for

MD9.6 (black), MD9.7 (red), and MD9.10 (green) 3169.7 Atomic pair distance fluctuation in simulation of MD9.7 3179.8 (-)-Sparteine conformations 3229.9 Atomic pair distance fluctuation in MD simulation of MD9.9 3239.10 Atomic pair distance fluctuation in MD simulation of MD9.10 325

9.11 Relative potential energy fluctuation during CPMD simulations

CPMD9.7 (C5=OH, black), CPMD9.8 (C5=NH2, red), CPMD9.9

(C5=C6H5CH2OCH2O-, green) and CPMD9.10 (C5=C5NH4CH2O-,

blue) 328

9.12 Atomic pair distance fluctuation in CPMD simulations for (a) CPMD9.7,

(b) CPMD9.8, (c) CPMD9.9, and (d) CPMD9.10 330

A.1 Thermodynamic Cycle for Binding Free Energy Calculation of FKBP12

and Probe Peptides 362A.2 Time Correlation Analysis of Four Complexes 362A.3 Decomposition of binding free energies to single residue in ligand

peptides based on separated MD trajectories 363

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A.4 Decomposition of Binding Free Energies to Single Residue from

FKBP12 (Residue37 to 72) based on Separated Trajectories 364

A.5 Decomposition of Binding Free Energies to Single Residue from

FKBP12 (Residue73~107) based on Separated Trajectories 365

B.1 Reaction mechanism for carboxylate migration through MICAp1 at

B.8 Mechanism for deprotonation pathway of MICAp1 carboxylate

migration product by histidine at B3LYP/6-31+G(d) level of theory 386

B.9 Mechanism for carboxyl group migration pathway with the complete

N5-CAIR structure, and including the ribose-5-phosphate unit at

B3LYP/6-31+G(d) level of theory 387C.1 Reaction mechanism for carboxylate migration through N5-CAIR 011-1

and CIAR 011-1 at B3LYP/6-31+G(d) level of theory 399

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C.2 Reaction mechanism for carboxylate migration through N5-CAIR 011-2

and CIAR 111-2 at B3LYP/6-31+G(d) level of theory 402D.1 Overlap of first 20 conformers of 7.2 from Monte Carlo searches 404

D.2 Two conformers of 7.2 with highest RMSD values with respect to global

minimum from Monte Carlo search 404D.3 Right-handed helix with negative dihedral angle (-22°) 405D.4 Dihedral Angle of on the one end of 7.1 406D.5 The distribution of distance between two carbons at the other end of 7.1 406

D.6 The distribution of distances between two carbons at two ends of 7.2 407

D.7 Four representative conformers from clustering analysis of replica 0 in

REMD simulation of 7.1 408E.1 Atomic Pair Distance Fluctuation in Simulation of MD8 (C5=NH2) 411

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CHAPTER 1

INTRODUCTION

Modern chemistry is often exemplified by the experimental study of matter at the atomic and molecular scales Computational chemistry is often used to complement such experimental studies so as to enhance one’s knowledge of the entire chemical system Also, due to the constantly increasing power of modern computer systems, computational chemistry methods are routinely used to research simple and sometimes complex systems

in chemistry Indeed, computational chemistry has established itself as a significant discipline of modern chemistry, and many chemical systems can be studied

sub-computationally so as to provide insights when experimental methods are cost prohibitive

or inconclusive for details at the molecular level In this dissertation, several chemical and biological systems were studied by appropriate computational methods, depending

on the nature of the properties studied In the next several sections, I will outline the general areas which were explored in this dissertation by the use of modern

computational methods in a diverse set of chemical and biological problems

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1.1 TRPC Ion Channels

Transient receptor potential-canonical (TRPC) channels are members of the mammalian transient receptor potential (TRP) channel superfamily of cation channels

These TRPC channels are mammalian homologues of Drosophila TRPC and TRPL

channels.1 Using sequence homology as criteria, the 28 members of the mammalian TRP family can be subdivided into seven subfamilies.2 The TRPC subfamily comprises seven members (TRPC1-C7) This subfamily is the most closely related to the original

Drosophila TRP and TRPL channels Based on sequence similarities, the seven members

of the TRPC subfamily can be further subdivided into four groups: (a) TRPC1; (b)

TRPC2; (c) TRPC3, 6, and 7; and (d) TRPC4 and 5 TRPC subfamilies share some structural properties For example, each member is a peptide that transverses the

membrane six times, with the N- and C-terminals located in the cytoplasm.3 And a proline-rich motif is located downstream of the last transmembrane domain of TRPCs Recently, it was shown that the ‘immunophilins’ FKBP52 and FKBP12 bind to TRPCs through this proline-rich domain during the regulation process of these ion channels.4 Specifically, TRPC1, TRPC4, and TRPC5 bind immunophilin FKBP52; and TRPC3, TRPC6, and TRPC7 bind immunophilin FKBP12

It is known that the activation and inactivation of TRPC6 channels is regulated by phosphorylation, by protein kinase C (PKC) negatively regulating the channels, and by either CaM kinase II or the nonreceptor tyrosine kinase Fyn positively regulating the channels Shi et al showed that Ca2+ entry activated by the DAG analogue OAG is

attenuated by either a calmodulin (CaM) antagonist, which is a CaM kinase II inhibitor,

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could be phosphorylated by Fyn, following epidermal growth factor (EGF).6

Immunophilins are peptidyl prolyl cis-trans isomerases that recognize specific XP

dipeptides in their target proteins and act as immunosuppressants.7 They bind strongly to the immunosuppressant drugs cyclosporin A, FK506, and rapamycin.8, 9 Cyclosporin A binds to members of the cyclophilin family, whereas FK506 and rapamycin bind to the family of FK506 binding proteins (FKBPs) The FKBPs could bind with two major intracellular calcium channels: the ryanodine receptor and the inositol 1,4,5-trisphosphate receptor (IP3R).10 In both cases, FKBP12 is tightly associated with the channels, and dissociation of FKBP12 from either channel perturbs the calcium flux.11,12 13 14, , The

binding between FKBPs and TRPCs was also found in Drosophila TRP, where FKBP59

binds the conserved proline-rich dipeptide (701LPPPFNVLPSVK709) at the C-terminal region close to the last transmembrane domain.15 This sequence is conserved in all TRPCs

Schilling’s group recently reported that TRPC1, TRPC4, and TRPC5 bind

immunophilin FKBP52, and TRPC3, TRPC6, and TRPC7 bind immunophilin FKBP12 Disruption of the FKBP12 binding delayed cation entry which is mediated by the TRPC channels This suggests that the binding of immunophilin plays a role in TRPC channel activation Recently, Kim and Saffen used coimmunoprecipitation to show that FKBP12

is a component of a TRPC6-centered protein complex, which rapidly forms following activation of endogenous M1 mAChR in neuronal PC12D cells.16 The structure of the TRPC6 and FKBP12 binding sites is illustrated in Figure 1.1, and FKBP12 plays a central role in the regulation of the TRPC6 channel Computational modeling was used

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to better understand how TRPC6 channels bind FKBP12 and how phosphorylation affects this process These results will be discussed in Chapter 3

FKPB12

Figure 1.1 Structure of TRPC6 Channel and FKBP12 Binding Site

1.2 N 5 -CAIR Mutase Mechanism in the Purine Biosynthesis Pathway

Purine is a heterocyclic aromatic organic compound: the basic structure of purine consists of a pyrimidine ring fused with an imidazole ring (Figure 1.2) Purine and its derivatives (normally called purines) are important molecules in biological systems; adenine and guanine, two bases in nucleic acids, are purines

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N

N H N

N N

N H N

NH2

N HN

N H N O

H2N1

Figure 1.2 Structures of purine, adenine and guanine

Almost all biological systems can synthesize purines de novo and from

degradation products of nucleic acids.17 When synthesized de novo, different parts of a

given purine ring originate from different sources: the N1 arises from the amine group

of the amino acid aspartate; C2 and C8 originate from formate (HCO3-); N3 and N9 are contributed by the amine group of the amino acid glutamine; C4, C5 and N7 are derived from the amino acid glycine; and C6 comes from HCO3- or CO2 (Figure 1.3)

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C

N

C N

C C

Aspartate

amineFormate

Formate

Glutamine amine

Figure 1.3 Biosynthetic sources of purine ring atoms.18

Approximately fifteen enzymes are involved in the de novo biosynthesis of purine

nucleotides In eukaryotes, many of these enzymes exist as multifunctional proteins Due to the importance of the products of this pathway, the imbalances in purine

regulation are related to numerous disorders, such as trisomy 21 (Down’s syndrome),19the neurological disorder Lesch-Nyhan syndrome,20 gout,21 and urinary and bladder stones.22 Several types of cancer cells have elevated levels of the de novo purine

enzymes.23,24 25, Therefore, these enzymes are targets for chemotherapeutic drug

discovery.26, 27 28 29 30 , , ,

The enzymatic reactions and intermediates along the pathway were largely

characterized by Buchanan and co-workers in the 1950s and 1960s.31 Recently, several

new enzyme activities along the de novo purine biosynthetic pathway have been

discovered For example, the mechanism of CAIR formation, catalyzed by PurK and

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Enzymes which catalyze the carboxylation of 5-aminoimidazole have been

investigated from a variety of prokaryotic and eukaryotic sources One enzyme,

previously designated as AIR carboxylase, has been proposed to catalyze the conversion

of AIR and bicarbonate or CO2 to CAIR However, two separate gene products,

designated PurE and PurK, are required for reactivity in E Coli.33,34 35, Stubbe and workers showed that PurE and PurK each possess independent catalytic activities.36 PurK catalyzes the conversion of AIR (in the presence of HCO3- and ATP) to the N5-carboxyaminoimidazole ribonucleotide (N5-CAIR), along with the byproducts ADP and

co-Pi PurE catalyzes the reversible conversion of N5-CAIR and CAIR Both chicken and human PurEs are found as part of a bifunctional protein which also carries

phosphoribosylaminoimidazole-succinocarboxamide (SAICAR) synthesis function However, the genetic homologues with PurK have not been found in these systems In other words, PurK is not a gene product in these organisms and is not required by these organisms in their purine biosynthetic pathways

This divergence of purine biosynthesis between prokaryotes and eukaryotes provides a novel target for drug design Inhibitors could be designed based on the

structural differences among these enzymes to selectively inhibit the functions of PurK or PurE in prokaryotes However, the reaction mechanisms of these enzymes still need to

be determined to facilitate rational structure-based drug design Fortunately, the crystal

structure of PurE in E coli is available from the Protein Data Bank (PDB).37 While the reaction mechanism of this enzyme has not been elucidated completely by experiments, these three-dimensional protein structures and the relevant enzymatic mechanism are

Tiêu đề	Computational studies to understand molecular regulation of the TRPC6 calcium channel, the mechanism of purine biosynthesis, and the folding of azobenzene oligomers
Tác giả	Peng Tao
Người hướng dẫn	Christopher M. Hadad, Advisor, Russell M. Pitzer, James V. Coe
Trường học	The Ohio State University
Chuyên ngành	Chemistry
Thể loại	Dissertation
Năm xuất bản	2007
Thành phố	Ann Arbor

Định dạng
Số trang	500
Dung lượng	14,96 MB