STRUCTURAL AND EQUILIBRIUM UNFOLDING STUDIES OF SAM DOMAIN OF DLC1 BY NMR SPECTROSCOPY YANG SHUAI NATIONAL UNIVERSITY OF SINGAPORE STRUCTURAL AND EQUILIBRIUM UNFOLDING STUDIES OF SAM DOMAIN OF DLC1 BY NMR SPECTROSCOPY THESIS BY YANG SHUAI SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2009 ABSTRACT Deleted in liver cancer-1 (DLC1) is found to be deleted in many primary human tumors and in human hepatocellular carcinoma (HCC) cell lines, which suggests that it is a tumor suppressor gene for these cancers. The DLC1 cDNA encodes a 1091amino acid protein which has a sterile alpha motif (SAM) domain at its N-terminus. The SAM domain of DLC1 (henceforth called DLC1-SAM) is the subject of this dissertation. SAM domain is a protein-protein interaction module of ~ 70 amino acid residues that can be found in many proteins the functions of which range from signal transduction to transcriptional repression. SAM domains are known to interact with various biomolecules, such as proteins, RNAs and even lipid. DLC1-SAM shares very low sequence identity with other SAM domains. We have determined the solution structure of DLC1-SAM using triple resonance NMR techniques. The overall 3D structure is similar to those of other SAM family members. However, DLC1-SAM consists of only four helices, instead of the five helices that are usually found in almost all other SAM domains. Additionally, the orientation of helices in the DLC1-SAM structure is different from that of other SAM domains. The solution structure of DLC1-SAM provides a basis for the determination of potential residues that are involved in interactions with a novel binding partner, EF1A1, of the SAM superfamily. The solution structure of DLC1-SAM as well as the resonance assignment of the native DLC1-SAM is the prerequisite for the study of the equilibrium unfolding of DLC1-SAM. i We have studied the urea-induced unfolding of DLC1-SAM by various biophysical methods, such as CD, fluorescence emission spectroscopy and NMR. The unfolding curves obtained from CD and tryptophan intrinsic fluorescence emission coincided within experimental error. It seemed that the unfolding of DLC1-SAM followed a simple two-state mechanism， but the NMR data suggested a different mechanism. For most residues with resolved resonances of the native and denatured states in the entire range of urea concentrations, there is a pronounced lag between the disappearing population of the native species and the appearing population of the denatured species. The sum of the populations of both native and denatured forms is not equal to unity in the transition zone, suggesting that at least one intermediate state is involved in the equilibrium unfolding. The equilibrium unfolding intermediate is confirmed not to be large aggregates by analytical ultracentrifugation experiments, and it might have fluorescent properties similar to those of the denatured state. Analysis of the free energy values for different residues shows that in the transition from the native state to non-native states, the C-terminal helix is somewhat more stable than the other parts of the protein, whereas in the transition from the native and intermediate states to the denatured state, the stabilities of different residues are similar except for the region surrounding residues D37 – F40 which has lower stability and is more readily denatured at high urea concentrations. Analysis of the midpoints of the transitions shows that the unfolding of the native state and formation of the denatured state are not cooperative and the unfolding of a few residues seems to follow a two-state mechanism. ii DEDICATION This dissertation is dedicated to my beloved parents, Yang Yufang and Ma Xiurong, for their love and endless support. iii ACKNOWLEDGEMENTS I am very appreciative of the good camaraderie and academic guidance that I have enjoyed in NUS in the last few years. I would like to thank my research supervisor, Associate Professor Yang Daiwen, for excellent ideas and scientific guidance. I also thank Dr Yang for imparting his logical approach to scientific research, his attention to details. I would like to thank Professors Thorsten Wohland and Mok Yu-Keung, Henry for their guidance as my graduate committee. I have benefited greatly from their scientific expertise. Thanks to everyone in Yang’s lab with whom I had a chance to interact. The combination of scientists with diverse backgrounds has made it a tremendous place to learn. In particular, I would like to thank Dr. Zhang Jingfeng for teaching me how molecular biology works in our lab and the basics of chromatographic and biophysical methods. Thanks to Dr. Xu Yingqi, Dr. Lin Zhi, Dr. Zhang Xu and Zheng Yu for helping me with the theoretical and practical basics of protein NMR spectroscopy and structure calculation. Thanks to everyone else in our lab at NUS including Sui Xiaogang, Balakrishna Chandrababu Karthik, Justin J. Joseph Gnanakkan, Meng Dan, Yong Yee Heng and Dr. K. P. Manoharan. Thanks to Dr. Fan Jingsong for all the NMR trainings and his kind assistance in NMR experiments. Thanks to Dr. Mok for the over-expression vector used in this work. Thanks to him and people in his lab including Dr. Zhang Yonghong, Xu Xingfu, Tan Yih iv Wan, Yvonne, Krishna Moorthy Janarthanan, Dr. Chan Siew Leong, Dr. Chiradip Chatterjee for helpful discussions in weekly meetings. Thanks to Associate Professor Low Boon Chuan and Zhong Dandan for their help as collaborators. Thanks to Assistant Professor Liang Zhao-Xun, Dr. Nikolay Korolev and Abdollah Allahverdi in Nanyang Technological University and Dr. Noble in Institute of Molecular and Cell Biology for their kind help in analytical ultracentrifuge experiments. Financially I was supported by the NUS research scholarship. I also benefited from SBPR financial support, because of which, I was fortunate to have the time to carry out experiments that I found worthwhile. Thanks to all the people in NUS that make my research work run so smoothly. Thanks to my mom and dad for being very encouraging and providing me with everything that I have needed since I was a baby. I have really enjoyed my last few years here at NUS thanks in large part to my friends. I am very grateful for the time that we get to share. Thanks to Minfen and Xiaogang. v TABLE OF CONTENTS ABSTRACT DEDICATION ACKNOWLEDGEMENTS LIST OF TABLES LIST OF FIGURES CHAPTER INTRODUCTION 1.1 Biological context 1.1.1 DLC1 gene and its biological functions 1.1.2 SAM domain and its biological functions 1.1.2.1 SAM domain-protein interaction 1.1.2.2 SAM domain-RNA interaction 1.1.2.3 SAM domain-lipid interaction 1.1.3 Structures of SAM domains 1.2 Protein structure determination by NMR spectroscopy 1.2.1 Fundamentals of NMR spectroscopy 1.2.1.1 NMR phenomenon 1.2.1.2 Basic NMR parameters 1.2.1.2.1 Chemical shift 1.2.1.2.2 J-coupling 1.2.1.2.3 Nuclear Overhauser Effect (NOE) 1.2.1.2.4 Chemical exchange 1.2.1.2.5 Relaxation 1.2.2 The advantages and limitation of NMR structural studies 1.2.3 General strategy of NMR structure determination 1.2.3.1 Sample preparation 1.2.3.2 Recording NMR spectra 1.2.3.3 Resonance assignments 1.2.3.4 Restraint collection 1.2.3.5 Structure calculation and refinement 1.2.3.6 Structure evaluation 1.3 Protein folding studies 1.3.1 Overview of Protein Folding Theories 1.3.1.1 Anfinsen’s dogma and Levinthal paradox 1.3.1.2 The “classical view” and the “new view” of protein folding 1.3.2 Equilibrium unfolding of proteins 1.3.2.1 Theoretical background 1.3.2.2 Protein denaturation induced by denaturant 1.3.2.3 Three main spectroscopic techniques used for protein folding studies 1.3.2.3.1 Circular dichroism (CD) spectroscopy vi i iii iv ix x 1 5 10 10 11 12 12 13 14 14 16 17 18 18 20 21 23 24 25 26 27 27 28 31 31 34 38 38 1.3.2.3.2 Fluorescence emission spectroscopy 1.3.2.3.3 NMR spectroscopy 1.4 Scope of research and outline of the thesis CHAPTER MATERIALS AND METHODS 2.1 Media 2.2 Expression vector construction 2.3 Expression and purification of DLC1-SAM 2.3.1 Expression of DLC1-SAM in E. coli 2.3.2 Purification of DLC1-SAM 2.4 Dynamic lights scattering (DLS) 2.5 NMR experiments and structure calculation 2.6 Structure-based alignment and structural comparison 2.7 Biophysical experiments for unfolding studies 2.7.1 Sample preparation 2.7.2 Fluorescence emission spectroscopy: data acquisition 2.7.3 CD spectroscopy: data acquisition 2.7.4 Resonance assignment of denatured protein 2.7.5 NMR spectroscopy: data acquisition, processing and analysis 2.8 Sedimentation velocity: data acquisition and analysis CHAPTER RESULTS AND DISCUSSION 3.1 Expression and purification of DLC1-SAM 3.1.1 Expression and purification of SAM60 3.1.2 Expression and purification of SAM76 3.2 NMR resonance assignment of DLC1-SAM 3.2.1 Backbone resonance assignments of SAM60 and SAM76 3.2.2 Aliphatic sidechain resonance assignment 3.2.3 Aromatic sidechain resonance assignment 3.2.3.1 Aromatic sidechain resonance assignment of SAM60 3.2.3.2 Aromatic sidechain resonance assignment of SAM76 3.2.4 Secondary structure prediction by chemical shift index (CSI) 3.2.5 NOE assignment 3.3 Solution structure of DLC1-SAM 3.3.1 NMR structure determination and description 3.3.2 Structure comparison between DLC1-SAM and other SAM domains 3.3.3 No self-association in DLC1-SAM 3.3.4 Prediction of possible binding site on the surface of DLC1-SAM 3.4 Equilibrium unfolding studies of DLC1-SAM60 3.4.1 Stabilities of SAM76 and SAM60 3.4.2 Urea-induced equilibrium unfolding followed by fluorescence and CD spectroscopy 3.4.2.1 CD and fluorescence spectra of SAM60 3.4.2.2 Unfolding curves obtained from fluorescence and CD spectroscopy coincide vii 39 41 50 51 51 52 52 53 54 54 57 57 57 58 58 59 59 63 64 64 66 67 67 70 70 70 75 79 79 85 85 89 96 100 102 102 104 104 106 3.4.3 Urea-induced equilibrium unfolding of DLC1-SAM followed by NMR spectroscopy 3.4.3.1 Resonance assignment of the denatured state 3.4.3.2 Urea-induced equilibrium unfolding monitored by NMR 3.4.3.3 Changes in the relaxation behavior of amide groups and correction of cross-peak volumes 3.4.3.4 Unfolding equilibrium intermediate revealed by NMR spectroscopy 3.4.3.5 Study of the unfolding process in a residue-specific way 3.4.4 No aggregation for the equilibrium unfolding intermediate: the sedimentation velocity studies 3.4.5 The properties of the equilibrium unfolding intermediate 3.4.6 Equilibrium unfolding of different SAM domains CHAPTER CONCLUSION AND FUTURE WORK REFERENCES ABBREVIATIONS viii 111 111 116 120 127 131 141 147 149 151 153 167 residual structures in the denatured state might be the initialization site of the folding of DLC1-SAM. In the future, the residual structures should be studied to a further extent. We should get more NMR derived parameters for the denatured DLC1-SAM and check Cα and Cβ chemical shift deviations from those of the random coil, and try to combine these observations to obtain information about residual structures. If possible, we should study the kinetic unfolding of DLC1-SAM and combine the detailed kinetic data with the equilibrium results in this thesis to further illustrate whether a folding intermediate is present or not during the (un)folding of DLC1-SAM. To summarize, the DLC1-SAM is a special member of the SAM domain super family as it has a solution structure that is unique among SAM domain structures. Good NMR properties of DLC1-SAM make it a very good model to study equilibrium unfolding of small proteins with a similar fold. 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J Cell Sci 122: 414-424. 166 ABBREVIATIONS AUC BAR BPTI CATH CD CREB CSI DG DG DLC1 DLS DQF-COSY DSC DTT EF1A1 EH Eph GAP GndHCl HCC HSQC INEPT IPTG ML NMR NOE NOESY OD PAGE PCR Ph PHAT RMSD RNase A SAM SD SDS START TEL TOCSY alalytical ultracentrifugation bifunctional apoptosis regulator bovine pancreatic trypsin inhibitor class architecture topology homology circular dichroism cAMP response element binding protein chemical shift index distance geometry distance geometry deleted in liver cancer dynamic lights scattering double-quantum filtered correlation spectroscopy differential scanning calorimetry dithiothreitol eukaryotic elongation factor-1A1 end-helix erythropoietin producing hepatocellular receptor GTPase activating protein guanidine hydrochloride hepatocellular carcinoma heteronuclear single-quantum coherence Insensitive Nuclei Enhanced by Polarization Transfer isopropyl-β-D-thiogalactopyranoside mid-loop nuclear magnetic resonance nuclear Overhauser effect nuclear Overhauser effect spectroscopy optical density polyacrylamide gel electrophoresis polymerase chain reaction polyhomeotic pseudo HEAT analogous topology root-mean square deviation ribonuclease A sterile alpha motif standard deviation sodium dodecyl sulfate StAR-related lipid-transfer translocation Ets leukemia total correlation spectroscopy 167 [...]... structure of DLC1 -SAM 88 Figure 3.13 Sequence alignment of DLC1 -SAM and other representative SAM domains with known structures 90 x Figure 3.14 Structural differences DLC2 -SAM between DLC1 -SAM and 92 Figure 3.15 The structural comparison between DLC1 -SAM (red) and Vts1 -SAM (green) 95 Figure 3.16 The size distribution of SAM6 0 (a) and SAM7 6 (b) measured by DLS 97 Figure 3.17 The van der Waals surface of DLC1 -SAM, ... Resonance assignment of aromatic protons of SAM7 6 78 Figure 3.9 Prediction of secondary structure of (a) SAM6 0 and (b) SAM7 6 using chemical shift index 80 Figure 3.10 Plots of the number of assigned NOEs of (a) SAM6 0 and (b) SAM7 6 as a function of the range of NOEs and the residue numbers (lower panel), respectively 82 Figure 3.11 Sequential and medium-range NOEs of (a) SAM6 0 and (b) SAM7 6 83 Figure 3.12... comprehensive overview of all NMR studies that have been done in the past few years, but rather serves to give readers a general idea of the basics of NMR spectroscopy and the protein structure determination by NMR spectroscopy 1.2.1 Fundamentals of NMR spectroscopy Although different detecting techniques and probes are used in NMR spectrometers, the basic theory of NMR is common to all experiments and nuclei... Parameters of native (cm1, m1, ΔG10) and denatured (cm2, m2, 133-134 ΔG2u) unfolding curves ix LIST OF FIGURES Figure 1.1 Ribbon diagrams of the structures of representative SAM domains 7 The flowchart of protein structure determination by NMR spectroscopy 19 Size-exclusion chromatograms of (a) SAM6 0 and (b) SAM7 6 65 Cα, Cβ connectivity for a stretch of residues from P29 to A32 68 Superposition of 1H-15N... C-terminal helix 5 of hEphB2 -SAM is much longer than that of the mEts1 -SAM, possibly because the C-terminal helix 5 of hEphB2 -SAM may play an important role in its self-association (Stapleton et al 1999; Thanos et al 1999) In addition, helix 2 is present in most SAM domains, but not in the SAM domains of Ets-1 and TEL 6 Figure 1.1 Ribbon diagrams of the structures of representative SAM domains SAM domains from... functions specific to each SAM domain The structure of DLC1 -SAM will expand our 8 view on the structure and biological functions of SAM domains 9 1.2 Protein structure determination by NMR spectroscopy Nuclear magnetic resonance spectroscopy, most commonly known as NMR spectroscopy, is the technique which studies magnetic properties of certain nuclei, such as 1H, 13 C, 15 N, 19 F and 31 P, etc in a magnetic...LIST OF TABLES Table 3.1 Table 3.2 Table 3.3 Experimental restraints and structural statistics for 10 lowest-energy NMR structures of SAM7 6 86 Experimental restraints and structural statistics for 10 lowest-energy NMR structures of SAM6 0 87 The backbone RMS deviations between DLC1 -SAM and other representative SAM domains 94 Table 3.4 1 H and 15N chemical shifts for urea-denatured SAM7 6 ([urea]... Ramachander et al 2002) Some SAM domains show the ability to interact with non -SAM domain- containing proteins In addition to the ability to bind proteins, new functions of SAM domains are being discovered Recent studies found that the SAM domain of Smaug could bind RNA (Aviv et al 2003; Green et al 2003), while the SAM domain of p73 is involved in lipid binding (Barrera et al 2003) 1.1.2.1 SAM domain- protein... the p73 -SAM structure (Barrera et al 2003) 5 Taken together, SAM domain is a protein module with diverse functions However, for some SAM domains, such as SAM domain of DLC1, little is known about their biological functions and 3D structures Thus, it remains a major challenge for researchers to determine their structures and assign new functions to those SAM domains 1.1.3 Structures of SAM domains Despite... middle of the sequence of the proteins, and the end-helix (EH) surface located around the C-terminal helix Except for the homo-polymeric structures mentioned above, SAM domains of Ste4 and Byr2 were found to bind to each other to form a 3:1 Ste-LZ -SAM: Byr2 -SAM complex (Ramachander et al 2002) 3 In addition to SAM- SAM association, SAM domains also interact with non -SAM domain- containing proteins (Stein et . STRUCTURAL AND EQUILIBRIUM UNFOLDING STUDIES OF SAM DOMAIN OF DLC1 BY NMR SPECTROSCOPY YANG SHUAI NATIONAL UNIVERSITY OF SINGAPORE STRUCTURAL AND EQUILIBRIUM. Prediction of possible binding site on the surface of DLC1 -SAM 100 3.4 Equilibrium unfolding studies of DLC1 -SAM6 0 102 3.4.1 Stabilities of SAM7 6 and SAM6 0 102 3.4.2 Urea-induced equilibrium unfolding. AND EQUILIBRIUM UNFOLDING STUDIES OF SAM DOMAIN OF DLC1 BY NMR SPECTROSCOPY THESIS BY YANG SHUAI SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL