Dynamics of liver fatty acid binding protein

DYNAMICS OF LIVER FATTY ACID BINDING PROTEIN LONG DONG (B.Sc.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2010 ACKNOWLEDGEMENTS I would like to express my deepest gratitude to my supervisor, Associate Professor Yang Daiwen, for his persistent support, inspiration and guidance during the course of this research project Without his encouragement and effort, completion of this thesis would not have been possible Special thanks to Assistant Professor Mu Yuguang (Nanyang Technological University) for his kind guidance and support in my computational studies, as well as for allowing me to access the computational facility in NTU The helpful discussions with Dr Dai Liang (Indiana University, USA) on various simulation techniques are also acknowledged I would like to thank Associate Professor Martin J Scanlon (Monash University, Australia) for sharing his research experience and results on fatty acid binding proteins, and thank Mr Song Wei (Xiamen University, China) for sharing his experimental results on the interaction between liver fatty acid binding protein and various ligands The critical research comments from Associate Professor Henry Mok in the group meeting discussion and technical assistance from Dr Fan Jingsong are also acknowledged I Thanks to my colleagues, labmates and friends in Singapore, with whom I enjoyed a pleasant learning experience In particular, I would like to thank B C Karthik, Chen Shuting, Dai Xuhui, Iman Fahim Hameed, Jiang Ping, Li Weifeng, Li Yanfu, Lim Jack Wee, Dr Lin Zhi, Meng Dan, Wang Shujing, Dr Xu Weixin, Dr Xu Yingqi, Yong Yee Heng, Dr Zhang Jingfeng, and Zheng Yu Many thanks to my parents who have been always encouraging and supporting me for the choices I made in my life Finally, the NUS research scholarship, which supported my graduate research work, is gratefully acknowledged II TABLE OF CONTENTS ACKNOWLEDGEMENTS TABLE OF CONTENTS SUMMARY LIST OF FIGURES LIST OF TABLES LIST OF ABBREVIATIONS CHAPTER INTRODUCTION 1.1 Overview of the fatty acid binding protein family 1.2 Overview of liver fatty acid binding protein (LFABP) 1.3 Mechanisms of the FABP-ligands interaction 1.4 Aim of the current studies I III VI IX XII XIII CHAPTER LITERATURE REVIEW 2.1 Structural studies on fatty acid binding proteins: implication for ligand entry and exit mechanisms 10 2.1.1 Structural studies on intestinal fatty acid binding protein 10 2.1.1.1 Three dimensional structure of holo-intestinal fatty acid binding protein 11 2.1.1.2 Structural studies on apo-intestinal fatty acid binding protein 12 2.1.2 Structural studies on liver fatty acid binding protein 13 2.1.2.1 Crystallographic study on holo-liver fatty acid binding protein 14 2.1.2.2 Structural studies on apo-liver fatty acid binding protein 15 2.1.3 A brief summary 15 2.2 Characterization of the dynamics of fatty acid binding proteins 16 2.2.1 Fast dynamics of fatty acid binding proteins on the picosecond to nanosecond timescales 16 2.2.2 Slow dynamics of fatty acid binding proteins on the microsecond to millisecond timescale 18 2.2.3 Molecular dynamics simulation of fatty acid binding proteins 19 2.2.4 Summary of the previous dynamics studies and the aim of current studies 21 2.3 NMR relaxation in liquids 22 2.3.1 Theory of spin relaxation in liquids 22 2.3.1.1 The master equation 22 2.3.1.2 Relaxation mechanisms: DD, CSA, and relaxation interference 24 2.3.2 NMR relaxation parameters and model free formalism 25 2.3.3 Conformational/chemical exchange effects in NMR spectroscopy 27 2.3.4 Transverse relaxation dispersion experiment 32 2.4 Molecular dynamics simulation 34 2.4.1 Basic principles 34 2.4.2 Force field for biomolecular simulations 35 III 2.4.3 Limitations of MD simulation 37 CHAPTER NMR SAMPLE PREPARATION, RESONANCE ASSIGNMENT AND STRUCTURE CALCULATION OF LFABP 39 3.1 Materials and methods 40 3.1.1 Media 40 3.1.2 SDS-polyacrylamide gel (SDS-PAGE) electrophoresis 40 3.1.3 Expression and purification of LFABP 41 3.1.4 NMR experiments for structure determination of LFABP 43 3.1.4.1 4D time-shared 13C/15N, 13C/15N-edited NOESY 44 3.1.4.2 MQ-CCH-TOCSY 46 3.1.4.3 HNCA experiment 46 3.1.5 Resonance assignment 47 3.1.5.1 Sequential assignment 47 3.1.5.2 Sidechain assignment 48 3.1.5.3 NOE assignments and structure calculation 48 3.2 Results and Discussion 49 3.2.1 Sample preparation of LFABP 49 3.2.2 NMR assignment of LFABP 54 3.2.2.1 Sequential assignment 54 3.2.2.2 Sidechain and NOE assignment 54 3.2.3 Structure calculation 58 3.3 Conclusion 63 CHAPTER PROBING SLOW MOTIONS OF LFABP ON MILLISECOND TIMESCALES 64 4.1 A general overview 65 4.2 Accurately probing millisecond timescale dynamics: The theory and method 65 4.2.1 Introduction: the theoretical background of the existing problem 66 4.2.2 Results and Discussion 67 4.2.2.1 Numerical optimization of the phase cycling scheme for relaxation dispersion experiment 67 4.2.2.2 NMR experimental evaluation 78 4.2.2.2.1 Methods and materials 78 4.2.2.2.2 Results and discussion 79 4.3 Probing slow dynamics of LFABP: A test of the assumptive model 83 4.3.1 Methods and Materials 83 4.3.2 Results and Discussion 85 4.3.2.1 An assumptive model 85 4.3.2.2 Intrinsic millisecond timescale dynamics of apo-LFABP 86 4.3.2.3 ANS binding studied by NMR titration 90 4.3.2.4 Kinetic rates of ANS binding at the high affinity site 92 4.3.2.5 Kinetic rates of ANS binding at the low affinity site 94 4.3.2.6 Chemical shift perturbation pattern an implication for the nature of the minor state 97 IV 4.3.3 Conclusion 100 CHAPTER BUFFER INTERFERENCE WITH PROTEIN DYNAMICS: A CASE STUDY ON LFABP 101 5.1 Introduction 102 5.2 Methods and materials 104 5.3 Results and discussion 106 5.3.1 MES binding inducing chemical shift perturbation 106 5.3.2 Effects of MES binding on protein dynamics on the picosecond to nanosecond timescale 110 5.3.3 Effects of MES binding on protein dynamics on the microsecond to millisecond timescale 112 5.3.4 Commonality of buffer interference with protein dynamics 114 5.4 Conclusion 117 CHAPTER MOLECULAR DYNAMICS SIMULATION OF LIGAND DISSOCIATION FROM LFABP 118 6.1 Introduction 119 6.2 Methods 119 6.2.1 Molecular dynamics simulation 119 6.2.2 Parameters for random expulsion simulation 121 6.2.3 Identification of residues constituting the portals 123 6.3 Results and Discussion 123 6.3.1 Dissociation of OLA128 from holo-LFABP 123 6.3.2 Dissociation of OLA129 from holo-LFABP 126 6.3.3 Residues constituting individual portals 130 6.3.4 Root mean squared fluctuations (RMSF) during the dissociations 133 6.3.5 Which portal does OLA129 dissociate from when OLA128 still binds the protein? 135 6.3.5 Dissociation of 1,8-ANS from LFABP 137 6.3.6 Comparative study between intestinal FABP and liver FABP 139 6.4 Conclusion 141 CHAPTER GENERAL CONCLUSIONS 142 REFERENCES 147 V SUMMARY Over a decade, scientists have been attempting to know more about the conformational dynamics of fatty acid binding proteins (FABPs), in order to answer the puzzling question – how ligands could access the internalized binding site(s) of FABPs Despite numerous efforts made in this field, the appreciation of this question is still relatively poor nowadays In the current study, we continued the effort to explore the dynamical properties of liver fatty acid binding protein (LFABP) using NMR spectroscopy and MD simulation techniques, aiming at advancing our knowledge on this interesting topic The microsecond to millisecond timescale dynamics of FABPs was historically hypothesized to represent a dynamical equilibrium between the “open” and “closed” states, regulating the ligand entry/exit processes Despite the potential significance, the validity of this hypothesis has not yet been demonstrated In the current study, the slow dynamics of LFABP was quantitatively characterized using relaxation dispersion NMR spectroscopy, which shows that LFABP is indeed highly flexible on the millisecond timescales In order to further examine the hypothetical role of the millisecond dynamics of LFABP, the potential correlation between slow dynamics and ligand entry/exit processes was modeled and evaluated by analyzing the kinetic rates of LFABP-ANS interaction The experimental result demonstrates that the intrinsic millisecond dynamics of LFABP, somewhat disappointedly, does not represent a critical conformational reorganization required for ligand entry due to the VI contradiction of timescales, but implies that it may represent a dynamical equilibrium between the apo-state and a state resembling the singly-bound conformation Analysis of the kinetic rates of the ligand association shows that the ligand-entry related dynamics could occur on the microsecond or sub-microsecond timescales, which is much faster than previously assumed Despite fast advancement of experimental techniques for exploring protein dynamics, direct visualization of ligand entry/exit processes which potentially involves multiple transient steps is still formidable nowadays In silico simulation, thus, provides a good alternative way to investigate such dynamical details However, the ligand exit/entry is a slow event which could hardly be accessed by standard MD simulations In order to overcome this problem, random expulsion simulation, which accelerates ligand motions with a randomly oriented external force, was applied to investigate the ligand dissociation processes (in Chapter 6) Different ligand egress routes were identified for LFABP in this work, which furthered our understanding on the protein-ligand interplay Future mechanistic studies on the ligand release and uptake would benefit from the experimental and computational studies shown in this thesis As a fortuitous discovery during our experimental studies, the millisecond timescale dynamics of LFABP was found to be perturbed by the presence of buffer agents Although not being our initial aim, we characterized the amplitude of such buffer perturbation to the slow motions of LFABP (in Chapter 5) This case study offers an VII example of how the biophysical properties of proteins could be influenced by buffer molecules, which would deserve the attention of scientists in the in vitro manipulation of protein molecules VIII LIST OF FIGURES Figure 1.1.1 Three-dimensional structures of FABPs Figure 2.3.1 Lineshapes of two-state chemcial exchange 31 Figure 3.1.1 The pET-32a derived plasmid (pET-M) 42 Figure 3.1.2 Pulse sequence for recording 4D time-shared 13C/15N, 13C/15N-edited NOESY 45 Figure 3.2.1 Expression and purification of LFABP 50 Figure 3.2.2 15N-1H HSQC spectra of LFABP 51 Figure 3.2.3 Overlay of HSQC spectra of LFABP with and without His-tag 53 Figure 3.2.4 The chemical shift correlation of the HNCA, 4D NOESY, and MQCCH-TOCSY spectra 55 Figure 3.2.5 Sequential connectivity for a stretch of residues (T96-K98) 56 Figure 3.2.6 Backbone assignment of LFABP 57 Figure 3.2.7 Superimposition of ten NMR conformers 60 Figure 3.2.8 Ramachandran plot of LFABP 61 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protein 13 2.1.2.1... binding proteins 16 2.2.1 Fast dynamics of fatty acid binding proteins on the picosecond to nanosecond timescales 16 2.2.2 Slow dynamics of fatty acid binding proteins on the microsecond to millisecond