PROBING PROTEIN DYNAMICS AND PROTEINPROTEIN INTERACTION BY NMR SPECTROSCOPY SUI XIAOGANG A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2010 Dedicated to the fond Memories of my late grandmother ACKNOWLEDGEMENTS I would first like to express my deep gratitude to my adviser, Dr. Yang Daiwen. His sustained support, kindness and dedication as research adviser and enthusiastic teacher, have guided and helped me through my Ph.D. studies. I would like to thank the committee members: Dr. Song Jianxing and Dr. Jaenicke Stephan, for their help and useful suggestions. I am sincerely grateful to all the co-workers whom I had worked with over the years in Yang’s group, for always being helpful and for making the lab a pleasant and inspiring workplace. I will be forever grateful to my families. Without their unending and untiring support, I cannot even start this thesis. Finally, the research scholarship provided by NUS is gratefully acknowledged. I TABLE OF CONTENTS ACKNOWLEDGEMENTS I  TABLE OF CONTENTS . II  LIST OF ABBREVIATIONS V  LIST OF TABLES . VII  LIST OF FIGURES . VIII  SUMMARY . XI  CHAPTER 1  GENERAL INTRODUCTION 1  1.1  History of NMR . 3  1.2  Basics of NMR . 5  1.2.1  Basic Theory 5  1.2.2  Relaxation Theory 7  1.2.2.1  Equation of Motion . 7  1.2.2.2  Semiclassical Relaxation Theory 8  1.2.3  1.2.3.1  1.2.4  Relaxation Mechanisms . 16  Intramolecular Dipole-Dipole Interaction . 16  Expressions for Spectral Densities . 18  1.2.4.1  Auto-correlated Dipolar(ij) Interaction . 18  1.2.4.2  Cross-correlated Dipole(ij)-Dipole(kl) Interaction . 18  1.2.5  Order Parameter . 20  1.2.5.1  Lipari and Szabo Approach 20  1.2.5.2  Order Parameter 22  II 1.3  Outlines of the thesis 23  CHAPTER 2  PROBING PROTEIN SIDE-CHAIN DYNAMICS . 24  2.1  Introduction 24  2.1.1  Backbone Dynamics and Side-Chain Dynamics . 25  2.1.2  Review of Protein Side-Chain Dynamics Studies . 27  2.1.3  Huntingtin Interaction Protein 32  2.2   2.2.1  Materials and Methods . 35  NMR Spectroscopy 35  2.2.1.1  Experiments 35  2.2.1.2  Data Procession . 36  2.2.1.3  Assignment of HIP2 Side-chain Methyl Groups 36  2.2.2  Extraction of Dynamics Parameters from Relaxation Data . 38  2.2.2.1  13 2.2.2.2  Spectral Density Function . 39  2.2.2.3  Rationality of S2 Calculation . 40  2.3  C Relaxation Data: R1 and Γ 38  Results and Discussion 43  2.3.1  Measurement of 13C Spin-Lattice Relaxation Rate 43  2.3.2  Measurement of Methyl Dipole-Dipole Cross-Correlated Relaxation Rate 47  2.3.3  Extraction of Dynamic Parameters 56  2.3.4  Application to HIP2 protein . 60  2.4  Conclusion . 70  CHAPTER 3  PROTEIN INTERFACE MAPPING 72  3.1  3.1.1  Introduction 72  Review of NMR Mapping Approaches . 74  III 3.1.2  Hemoglobin 76  3.1.3  Distance Driven Docking . 79  3.2  Materials and Methods . 80  3.2.1  3.2.1.1  NMR Spectroscopy 80  Data Procession . 80  3.2.2  Initial Relaxation Rate Measurement . 81  3.2.3  Derivation of the Relaxation Equation 83  3.2.4  Effective Distance Measurement . 85  3.2.5  Docking Study . 89  3.3  Results and Discussion 91  3.3.1  Initial Relaxation Rate Results . 91  3.3.2  Effective Distance Results . 95  3.3.3  Docking Results . 99  3.4  3.3.3.1  Hb A Dimer . 99  3.3.3.2  Hb A Tetramer 99  Conclusion . 106  CHAPTER 4  CONCLUSIONS AND FUTURE DIRECTIONS 107  4.1  Conclusions 107  4.2  Future Directions . 109  APPENDIX . 110  Appendix . 111  Appendix . 113  REFERENCE . 114  IV LIST OF ABBREVIATIONS This is a list of abbreviations in alphabetical order, to serve as a quick reference. AIRs Ambiguous Interaction Restraints ASA Accessible Surface Area BMR Bloch–Wangsness–Redfield Theory Da (kDa) Dalton (Kilodalton) DD Dipole-Dipole DR initial relaxation Rate Differences E1(s) Ubiquitin Activating Enzyme(s) E2(s) Ubiquitin Conjugating Enzyme(s) E3(s) Ubiquitin Ligase Enzyme(s) FID Free Induction Decay FT Fourier Transform HADDOCK High Ambiguity Driven biomolecular DOCKing HIP2 Huntingtin interaction protein V Hb A Human Adult Hemoglobin LS Lipari and Szabo NMR Nuclear Magnetic Resonance NOE nuclear Overhauser effect RF Radio Frequency UB/UBL Ubiquitin/Ubquitin-Like Protein VI LIST OF TABLES Table 2-1 Dynamics parameters of methyl groups in ubiquitin derived from spinlattice relaxation time T1 and dipole-dipole cross-correlated relaxation Γ . 55  Table 2-2 Dynamics parameters of methyl groups in HIP2 derived from spin-lattice relaxation time T1 and dipole-dipole cross-correlated relaxation Γ 65  Table 3-1 Effective distances (reff) of -chain of Hb A estimated from amide relaxation rates R1 and calculated from the R2 structure of Hb 98  Table 3-2 Hb A data to generate ambiguous interaction restraints (AIRs) 101  VII LIST OF FIGURES Figure 1-1 Redfield kite. 13  Figure 2-1 Sequence and secondary structure of HIP2 (PDB Code: 1YLA) 34  Figure 2-2 Contour plot of errors (in percentage) in the measured 13C{1H} NOE values as a function of τe and S2axis for methyl groups in a uniformly 13Clabeled protein . 42  Figure 2-3 Pulse sequence for the measurement of 13C T1 of methyl groups in uniformly 13C-labeled proteins 45  Figure 2-4 Contour plots of errors (in percentage) in the measured 13C R1 values as a function of τe and S2axis for methyl groups in a uniformly 13C-labeled protein with an overall correlation time of ns (a) or 15 ns (b) . 46  Figure 2-5 Pulse scheme for the measurement of dipole-dipole cross-correlated relaxation in methyl groups of uniformly 13C, 15N-labeled proteins . 48  Figure 2-6 Comparison of the values of X=1+x calculated from Equation 2-14 (•) and those estimated from Equation 2-15 and 2-16 (solid line, -) 51  Figure 2-7 Time dependence of the relative peak intensities of a number of representative methyl groups in ubiquitin . 54  Figure 2-8 Comparison of order parameters Saxis2 determined from 13C T1s and methyl dipole-dipole cross-correlated relaxation rates versus the values determined from 2H relaxation times T1 and T2 58  VIII Ernst, R. 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Journal of the American Chemical Society 119(29): 6711-6721. 137 [...]... system (Fields and Song 1989), affinity purification with MS/MS identification (Aebersold and Mann 2003), methods based on fluorescence technologies (Yan and Marriott 2004) and a variety of NMR spectroscopy approaches (Zuiderweg 2002) This thesis focuses on developing novel methods based on NMR relaxation techniques to study protein structure-function relationship and to reveal proteinprotein interactions... methods for mapping proteinprotein interaction interfaces XI In the second part of this thesis (Chapter 3), a new strategy to map proteinprotein interfaces on the basis of the dependence of NMR relaxation on proton density is presented This strategy needs two moderately deuterated samples in which the reporting protein in a protein- protein complex is 2H-,15N-labeled while the acceptor protein is either... important theories for liquid spin dynamics (Bloch 1956; Redfield 1957) 3 In 1964, Ernst and Anderson firstly obtained NMR spectrum by applying Fourier Transform (FT) of the free induction decay (FID) and demonstrated the improvement in signal to noise ratio over the continuous wave method (Ernst and Anderson 1966), which brought a new era of pulsed Fourier Transform NMR spectroscopy In 1971, Jeener proposed... proposed the idea of two dimensional (2D) NMR spectroscopy during a lecture, which was then realized by Aue et al (Aue et al 1976) The applications of 2D NMR spectroscopy led to an emergence of new methods and techniques In the following years, 2D NMR spectroscopy was rapidly applied on the detection of correlations between proton and carbon-13 spins (Maudsley and Ernst 1977; Maudsley et al 1977), the... (Wokaun and Ernst 1977a; Wokaun and Ernst 1977), investigations of chemical exchange (Jeener et al 1979; Meier and Ernst 1979), the detection of nuclear Overhauser effects (NOE) (Kumar et al 1980), and the determination of the first three dimensional protein structure by Wüthrich and coworker (Williamson et al 1985) With the availability of NMR probe techniques to record multiple frequencies, 2D NMR spectroscopy. .. cross-correlated relaxation and 13 C spin-lattice relaxation and those derived previously from 2H relaxation data, demonstrates the reliability and validity of this method Subsequently, the method was applied to the study of methyl dynamics of Huntingtin interacting protein 2 (HIP2) Currently, a variety of NMR based methods have been developed to identify protein- protein interaction sites, but they... review a general history and basic theories of NMR spectroscopy 2 1.1 History of NMR In 1890, prior to the discovery of NMR, Zeeman observed unusual behavior of certain nuclei in a magnetic field This phenomenon was later found to be due to nuclear spin (Zeeman 1897) After several decades, NMR was first discovered independently by Purcell et al and Bloch et al in 1946 Purcell observed NMR signal from Paraffin... Gutowsky and Pake discovered the huge potentials of NMR in the field of molecular structures (Gutowsky and Pake 1948; Gutowsky et al 1949) In 1950, Proctor and Yu discovered the phenomenon of the chemical shift (Proctor and Yu 1950) One year after, Gutowsky and McCall observed the J coupling effect (Gutowsky and McCall 1951) Meanwhile, Hahn discovered spin echoes and developed several other methods for probing. .. Mechanisms NMR relaxation is dominated by single or multiple mechanisms such as dipole-dipole interaction, chemical shift anisotropic interaction, quadrupolar interaction, scalar relaxation and more Intramolecular dipole-dipole interaction will be discussed here 1.2.3.1 Intramolecular Dipole-Dipole Interaction In a molecule, two spins which are close enough (≤ 10Å) experience significant dipolar interactions... of the techniques for protein structure determination at the atomic resolution It is the only currently available technique to reveal protein dynamics on per atom basis over a wide range of timescales in solution where physiological conditions can be imitated Although NMR experiments have been developed to characterize protein dynamics for different purposes, protein side-chain dynamics is still difficult . PROBING PROTEIN DYNAMICS AND PROTEIN- PROTEIN INTERACTION BY NMR SPECTROSCOPY SUI XIAOGANG A THESIS SUBMITTED. Dynamics and Side-Chain Dynamics 25 2.1.2 Review of Protein Side-Chain Dynamics Studies 27 2.1.3 Huntingtin Interaction Protein 2 32 2.2  Materials and Methods 35 2.2.1 NMR Spectroscopy 35 2.2.1.1. Lipari and Szabo Approach 20 1.2.5.2 Order Parameter 22 III 1.3 Outlines of the thesis 23 CHAPTER 2 PROBING PROTEIN SIDE-CHAIN DYNAMICS 24 2.1 Introduction 24 2.1.1 Backbone Dynamics and