Structural and equilibrium unfolding studies of sam domain of DLC1 by NMR spectroscopy

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. The exploration of the folding of DLC1-SAM should have a profound impact on the unfolding studies of proteins with a DLC1-SAM-like fold. 152 REFERENCES Anfinsen, C.B. 1973. Principles that govern the folding of protein chains. Science 181: 223-230. Anfinsen, C.B., Haber, E., Sela, M., and White, F.H., Jr. 1961. The kinetics of formation of native ribonuclease during oxidation of the reduced polypeptide chain. Proc Natl Acad Sci USA 47: 1309-1314. Arai, M., and Kuwajima, K. 2000. Role of the molten globule state in protein folding. Adv Protein Chem 53: 209-282. Arcus, V.L., Vuilleumier, S., Freund, S.M.V., Bycroft, M., and Fersht, A.R. 1995. A comparison of the pH, urea, and temperature-denatured states of barnase by heteronuclear NMR - implications for the initiation of protein-folding. J Mol Biol 254: 305-321. Aviv, T., Lin, Z., Lau, S., Rendl, L.M., Sicheri, F., and Smibert, C.A. 2003. The RNA-binding SAM domain of Smaug defines a new family of post-transcriptional regulators. Nat. Struct. Biol. 10: 614-621. Bagby, S., Go, S., Inouye, S., Ikura, M., and Chakrabartty, A. 1998. Equilibrium folding intermediates of a Greek key beta-barrel protein. J Mol Biol 276: 669-681. Bai, Y., Milne, J.S., Mayne, L., and Englander, S.W. 1993. Primary structure effects on peptide group hydrogen exchange. Proteins 17: 75-86. Baker, K.M., Wei, G., Schaffner, A.E., and Ostrowski, M.C. 2003. Ets-2 and components of mammalian SWI/SNF form a repressor complex that negatively regulates the BRCA1 promoter. J Biol Chem 278: 17876-17884. Baldwin, R.L. 2002. Making a network of hydrophobic clusters. Science 295: 1657-1658. Barbar, E., LiCata, V.J., Barany, G., and Woodward, C. 1997. Local fluctuations and global unfolding of partially folded BPTI detected by NMR. Biophys Chem 64: 45-57. Barrera, F.N., Garzon, M.T., Gomez, J., and Neira, J.L. 2002. Equilibrium unfolding of the C-terminal SAM domain of p73. Biochemistry 41: 5743-5753. 153 Barrera, F.N., Poveda, J.A., Gonzalez-Ros, J.M., and Neira, J.L. 2003. Binding of the C-terminal sterile alpha motif (SAM) domain of human p73 to lipid membranes. J Biol Chem 278: 46878-46885. Baum, J., Dobson, C.M., Evans, P.A., and Hanley, C. 1989. Characterization of a partly folded protein by NMR methods: studies on the molten globule state of guinea pig alpha-lactalbumin. Biochemistry 28: 7-13. Bhunia, A., Domadia, P.N., Xu, X., Gingras, R., Ni, F., and Bhattacharjya, S. 2008. Equilibrium unfolding of the dimeric SAM domain of MAPKKK Ste11 from the budding yeast: role of the interfacial residues in structural stability and binding. Biochemistry 47: 651-659. Blanco, F.J., Serrano, L., and Forman-Kay, J.D. 1998. High populations of non-native structures in the denatured state are compatible with the formation of the native folded state. J Mol Biol 284: 1153-1164. Bork, P., and Koonin, E.V. 1998. Predicting functions from protein sequences - where are the bottlenecks? Nat Genet 18: 313-318. Botelho, M.G., Gralle, M., Oliveira, C.L., Torriani, I., and Ferreira, S.T. 2003. Folding and stability of the extracellular domain of the human amyloid precursor protein. J Biol Chem 278: 34259-34267. Buevich, A.V., and Baum, J. 1999. Dynamics of unfolded proteins: incorporation of distributions of correlation times in the model free analysis of NMR relaxation data. J. Am. Chem. Soc. 121: 8671-8672. Case, D.A., Pearlman, D.A., Caldwell, J.W., III, T.E.C., Wang, J., Ross, W.S., Simmerling, C., Darden, T., Merz, K.M., Stanton, R.V., et al. 2002. AMBER 7. University of California, San Francisco, San Francisco, CA. Cavanagh, J. 2007. Protein NMR spectroscopy : principles and practice 2nd ed. Boston : Academic Press, Amsterdam. Chakrabarti, S.R., Sood, R., Ganguly, S., Bohlander, S., Shen, Z.Y., and Nucifora, G. 1999. Modulation of TEL transcription activity by interaction with the ubiquitin-conjugating enzyme UBC9. Proc Natl Acad Sci USA 96: 7467-7472. Chamberlain, A.K., and Marqusee, S. 1998. Molten globule unfolding monitored by hydrogen exchange in urea. Biochemistry 37: 1736-1742. 154 Chi, S.W., Ayed, A., and Arrowsmith, C.H. 1999. Solution structure of a conserved C-terminal domain of p73 with structural homology to the SAM domain. Embo J 18: 4438-4445. Cornilescu, G., Delaglio, F., and Bax, A. 1999. Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J Biomol NMR 13: 289-302. Daggett, V., and Fersht, A. 2003a. The present view of the mechanism of protein folding. Nat Rev Mol Cell Biol 4: 497-502. Daggett, V., and Fersht, A.R. 2003b. Is there a unifying mechanism for protein folding? Trends Biochem Sci 28: 18-25. De Prat Gay, G., Ruiz-Sanz, J., Neira, J.L., Itzhaki, L.S., and Fersht, A.R. 1995. Folding of a nascent polypeptide chain in vitro: cooperative formation of structure in a protein module. Proc Natl Acad Sci USA 92: 3683-3686. Delaglio, F., Grzesiek, S., Vuister, G.W., Zhu, G., Pfeifer, J., and Bax, A. 1995. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6: 277-293. DeLano, W.L. 2002. The PyMOL molecular graphics system DeLano Scientific, San Carlos, CA, USA. Dill, K.A., and Chan, H.S. 1997. From Levinthal to pathways to funnels. Nat Struct Biol 4: 10-19. Dobson, C.M., and Karplus, M. 1999. The fundamentals of protein folding: bringing together theory and experiment. Curr Opin Struct Biol 9: 92-101. Donaldson, L.W. Solution structure of the Ste11 SAM domain to be published. Dyson, H.J., and Wright, P.E. 2001. Nuclear magnetic resonance methods for elucidation of structure and dynamics in disordered states. In Nuclear Magnetic Resonance of Biological Macromolecules, Pt B, pp. 258-270. Eliezer, D., and Wright, P.E. 1996. Is apomyoglobin a molten globule? Structural characterization by NMR. J Mol Biol 263: 531-538. Eliezer, D., Yao, J., Dyson, H.J., and Wright, P.E. 1998. Structural and dynamic 155 characterization of partially folded states of apomyoglobin and implications for protein folding. Nat Struct Biol 5: 148 -155. Englander, S.W., and Kallenbach, N.R. 1984. Hydrogen exchange and structural dynamics of proteins and nucleic acids. Quart Rev Biophys 16: 521–655. Evans, J.N.S. 1995. Biomolecular NMR spectroscopy. New York: Oxford University Press, Oxford. Fei, J.W., Angel, P., Wei, Q.X., and de Villiers, E.M. 2006. TAp63 alpha indirectly regulates a cutaneous HPV promoter through complex formation with Jun family members. Oncogene 25: 3914-3923. Fersht, A.R. 1997. Nucleation mechanisms in protein folding. Curr Opin Struct Biol 7: 3-9. Foulds, C.E., Nelson, M.L., Blaszczak, A.G., and Graves, B.J. 2004. Ras/mitogen-activated protein kinase signaling activates Ets-1 and Ets-2 by CBP/p300 recruitment. Mol Cell Biol 24: 10954-10964. Fritz, R.D., and Radziwill, G. 2005. The scaffold protein CNK1 interacts with the angiotensin II type receptor. Biochem Biophys Res Commun 338: 1906-1912. Garcia, P., Serrano, L., Rico, M., and Bruix, M. 2002. An NMR view of the folding process of a CheY mutant at the residue level. Structure 10: 1173-1185. Goroncy, A. et al. NMR structure of sterile alpha motif (SAM) domain of Ephrin type-A receptor to be published. Green, J.B., Gardner, C.D., Wharton, R.P., and Aggarwal, A.K. 2003. RNA recognition via the SAM domain of Smaug. Mol. Cell 11: 1537-1548. Grimshaw, S.J., Mott, H.R., Stott, K.M., Nielsen, P.R., Evetts, K.A., Hopkins, L.J., Nietlispach, D., and Owen, D. 2004. Structure of the sterile alpha motif (SAM) domain of the Saccharomyces cerevisiae mitogen-activated protein kinase pathway-modulating protein STE50 and analysis of its interaction with the STE11 SAM. J Biol Chem 279: 2192-2201. Grimsley, J.K., Scholtz, J.M., Pace, C.N., and Wild, J.R. 1997. Organophosphorus hydrolase is a remarkably stable enzyme that unfolds through a homodimeric intermediate. Biochemistry 36: 14366-14374. 156 Güntert, P., Braun, W., and Wüthrich, K. 1991. Efficient computation of three-dimensional protein structures in solution from nuclear magnetic resonance data using the program DIANA and the supporting programs CALIBA, HABAS and GLOMSA. J Mol Biol 217: 517-530. Güntert, P., Mumenthaler, C., and Wüthrich, K. 1997. Torsion angle dynamics for NMR structure calculation with the new program DYANA. J Mol Biol 273: 283-298. Hackzell, A., Uramoto, H., Izumi, H., Kohno, K., and Funa, K. 2002. p73 independent of c-Myc represses transcription of platelet-derived growth factor beta-receptor through interaction with NF-Y. J Biol Chem 277: 39769-39776. Herrmann, T., Guntert, P., and Wüthrich, K. 2002. Protein NMR structure determination with automated NOE assignment using the new software CANDID and the torsion angle dynamics algorithm DYANA. J Mol Biol 319: 209-227. Hock, B., Bohme, B., Karn, T., Yamamoto, T., Kaibuchi, K., Holtrich, U., Holland, S., Pawson, T., Rubsamen-Waigmann, H., and Strebhardt, K. 1998. PDZ-domain-mediated interaction of the Eph-related receptor tyrosine kinase EphB3 and the ras-binding protein AF6 depends on the kinase activity of the receptor. Proc Natl Acad Sci USA 95: 9779-9784. Holm, L., and Sander, C. 1998. Touring protein fold space with Dali/FSSP. Nucleic Acids Res 26: 316-319. Homma, Y., and Emori, Y. 1995. A dual functional signal mediator showing rhogap and phospholipase c-delta stimulating activities. Embo J 14: 286-291. Hosoda, M., Ozaki, T., Miyazaki, K., Hayashi, S., Furuya, K., Watanabe, K., Nakagawa, T., Hanamoto, T., Todo, S., and Nakagawara, A. 2005. UFD2a mediates the proteasomal turnover of p73 without promoting p73 ubiquitination. Oncogene 24: 7156-7169. Huyghues-Despointes, B.M., Scholtz, J.M., and Pace, C.N. 1999. Protein conformational stabilities can be determined from hydrogen exchange rates. Nat Struct Biol 6: 910-912. Jackson, S.E. 1998. How small single-domain proteins fold? Fold Des 3: R81-91. Jackson, S.E., and Fersht, A.R. 1991. Folding of chymotrypsin inhibitor-2 .1. Evidence for a 2-state transition. Biochemistry 30: 10428-10435. 157 Jarymowycz, V.A., and Stone, M.J. 2006. Fast time scale dynamics of protein backbones: NMR relaxation methods, applications, and functional consequences. Chem Rev 106: 1624-1671. Johnson, B.A. 2004. Using NMRView to visualize and analyze the NMR spectra of macromolecules. Methods Mol Biol 278: 313-352. Johnson, P.E., and Donaldson, L.W. 2006. RNA recognition by the Vts1p SAM domain. Nat Struct Mol Biol 13: 177-178. Karplus, M., and Weaver, D.L. 1976. Protein-folding dynamics. Nature 260: 404-406. Kasten, M., and Giordano, A. 2001. Cdk10, a Cdc2-related kinase, associates with the Ets2 transcription factor and modulates its transactivation activity. Oncogene 20: 1832-1838. Kay, L., Keifer, P., and Saarinen, T. 1992. Pure absorption gradient enhanced heteronuclear single quantum correlation spectroscopy with improved sensitivity. J. Am. Chem. Soc. 114: 10663-10665. Kim, C.A. et al. Structural organization of a SAM domain co-polymer from the polycomb group proteins polyhomeotic and sex-comb-on-midleg to be published. Kim, C.A., and Bowie, J.U. 2003. SAM domains: uniform structure, diversity of function. Trends Biochem. Sci. 28: 625-628. Kim, C.A., Gingery, M., Pilpa, R.M., and Bowie, J.U. 2002. The SAM domain of polyhomeotic forms a helical polymer. Nat Struct Biol 9: 453-457. Kim, C.A., Phillips, M.L., Kim, W., Gingery, M., Tran, H.H., Robinson, M.A., Faham, S., and Bowie, J.U. 2001. Polymerization of the SAM domain of TEL in leukemogenesis and transcriptional repression. Embo J 20: 4173-4182. Kim, P.S., and Baldwin, R.L. 1982. Specific intermediates in the folding reactions of small proteins and the mechanism of protein folding. Annu Rev Biochem 51: 459-489. Klein-Seetharaman, J., Oikawa, M., Grimshaw, S.B., Wirmer, J., Duchardt, E., Ueda, T., Imoto, T., Smith, L.J., Dobson, C.M., and Schwalbe, H. 2002. Long-range interactions within a nonnative protein. Science 295: 1719-1722. 158 Koradi, R., Billeter, M., and Wüthrich, K. 1996. MOLMOL: A program for display and analysis of macromolecular structures. J. Mol. Graphics 14: 51-55. Kwan, J.J., and Donaldson, L.W. 2007. The NMR structure of the murine DLC2 SAM domain reveals a variant fold that is similar to a four-helix bundle. BMC Struct Biol 7. Ladokhin, A.S. 2000. Fluorescence spectroscopy in peptide and protein analysis. In Encyclopedia of Analytical Chemistry: Instrumentation and Applications. (ed. R.A. Meyers), pp. 5762 - 5779. Wiley, Chichester. Laskowski, R.A., Rullmann, J.A.C., MacArthur, M.W., Kaptein, R., and Thornton, J.M. 1996. AQUA and PROCHECK-NMR: Programs for checking the quality of protein structures solved by NMR. J Biomol NMR 8: 477-486. Latypov, R.F., Cheng, H., Roder, N.A., Zhang, J., and Roder, H. 2006. Structural characterization of an equilibrium unfolding intermediate in cytochrome c. J. Mol. Biol. 357: 1009-1025. Levinthal, C. 1968. Are there pathways for protein folding? J Chem Phys 65: 44-45. Li, C., Charlton, L.M., Lakkavaram, A., Seagle, C., Wang, G., Young, G.B., Macdonald, J.M., and Pielak, G.J. 2008. Differential dynamical effects of macromolecular crowding on an intrinsically disordered protein and a globular protein: implications for in-cell NMR spectroscopy. J Am Chem Soc 130: 6310-6311. Li, H., Fung, K.L., Jin, D.Y., Chung, S.M., Ching, Y.P., Ng, O.L., Sze, K.H., Ko, C.B., and Sun, H. 2007. Solution structures, dynamics, and lipid-binding of the sterile alpha-motif domain of the deleted in liver cancer 2. Proteins 67: 1154-1166. Logan, T.M., Theriault, Y., and Fesik, S.W. 1994. Structural characterization of the FK506 binding protein unfolded in urea and guanidine hydrochloride. J Mol Biol 236: 637-648. Matsuda, S., Harries, J.C., Viskaduraki, M., Troke, P.J.F., Kindle, K.B., Ryan, C., and Heery, D.M. 2004. A conserved alpha-helical motif mediates the binding of diverse nuclear proteins to the SRC1 interaction domain of CBP. J Biol Chem 279: 14055-14064. Mok, Y., Kay, C.M., Kay, L.E., and Forman-Kay, J.D. 1998. NOE data demonstrating a compact state for an SH3 domain under non-denaturing conditions. J Mol 159 Biol 289: 619-638. Mok, Y.K., Alonso, L.G., Lima, L.M., Bycroft, M., and de Prat-Gay, G. 2000. Folding of a dimeric beta-barrel: residual structure in the urea denatured state of the human papillomavirus E2 DNA binding domain. Protein Sci 9: 799-811. Myers, J.K., Pace, C.N., and Scholtz, J.M. 1995. Denaturant m values and heat capacity changes: relation to changes in accessible surface areas of protein unfolding. Protein Sci. 4: 2138-2148. Nagaya, H., Wada, I., Jia, Y.J., and Kanoh, H. 2002. Diacylglycerol kinase delta suppresses ER-to-Golgi traffic via its SAM and PH domains. Mol Biol Cell 13: 302-316. Ng, I.O.-l., Liang, Z.-d., Cao, L., and Lee, T.K.-w. 2000. DLC-1 is deleted in primary hepatocellular carcinoma and exerts inhibitory effects on the proliferation of hepatoma cell lines with deleted DLC-1. Cancer Res 60: 6581-6584. Nosoh, Y., and Sekiguchi, T. 1991. Protein stability and stabilization through protein engineering. E. Horwood, New York pp. 223. Orengo, C.A., Michie, A.D., Jones, S., Jones, D.T., Swindells, M.B., and Thornton, J.M. 1997. CATH--a hierarchic classification of protein domain structures. Structure 5: 1093-1108. Pace, C.N. 1975. The stability of globular proteins. CRC Crit Rev Biochem 3: 1-43. Pace, C.N. 1986. Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods Enzymol. 131: 266-280. Pace, C.N., Trevino, S., Prabhakaran, E., and Scholtz, J.M. 2004. Protein structure, stability and solubility in water and other solvents. Philos Trans R Soc Lond B Biol Sci 359: 1225-1234; discussion 1234-1225. Park, S.W., Durkin, M.E., Thorgeirsson, S.S., and Popescu, N.C. 2003. DNA variants of DLC-1, a candidate tumor suppressor gene in human hepatocellular carcinoma. Int J Oncol 23: 133-137. Pearl, F., Todd, A., Sillitoe, I., Dibley, M., Redfern, O., Lewis, T., Bennett, C., Marsden, R., Grant, A., Lee, D., et al. 2005. The CATH Domain Structure Database and related resources Gene3D and DHS provide comprehensive 160 domain family information for genome analysis. Nucleic Acids Res 33: D247-251. Ponting, C.P., and Aravind, L. 1999. START: a lipid binding domain in StAR, HD-ZIP and signalling proteins. Trends Biochem Sci 24: 130-132. Ptitsyn, O.B. 1987. Protein folding: hypotheses and experiments. J Protein Chem 6: 273-293. Ptitsyn, O.B. 1995. Molten globule and protein folding. Adv Protein Chem 47: 83-229. Purdie, N., and Swallows, K.A. 1989. Analytical applications of polarimetry, optical rotatory dispersion, and circular dichroism. Anal Chem 61: 77A-89A. Quezada, C.M., Schulman, B.A., Froggatt, J.J., Dobson, C.M., and Redfield, C. 2004. Local and global cooperativity in the human alpha-lactalbumin molten globule. J Mol Biol 338: 149-158. Ramachander, R., Kim, C.A., Phillips, M.L., Mackereth, C.D., Thanos, C.D., McIntosh, L.P., and Bowie, J.U. 2002. Oligomerization-dependent association of the SAM domains from Schizosaccharomyces pombe Byr2 and Ste4. J Biol Chem 277: 39585-39593. Ramboarina, S., and Redfield, C. 2003. Structural characterisation of the human alpha-lactalbumin molten globule at high temperature. J Mol Biol 330: 1177-1188. Redfield, C., Schulman, B.A., Milhollen, M.A., Kim, P.S., and Dobson, C.M. 1999. Alpha-lactalbumin forms a compact molten globule in the absence of disulfide bonds. Nat Struct Biol 6: 948-952. Roberts, J.D. 2000. ABCs of FT-NMR University Science Books, 2000, Sausalito, CA Sadqi, M., Lapidus, L.J., and Munoz, V. 2003. How fast is protein hydrophobic collapse? Proc Natl Acad Sci USA 100: 12117-12122. Santoro, M.M., and Bolen, D.W. 1988. Unfolding free energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl alpha-chymotrypsin using different denaturants. Biochemistry 27: 8063-8068. 161 Schellman, J.A. 1978. Solvent denaturation. Biopolymers 17: 1305–1322. Schuck, P. 2000. Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. Biophys. J. 78: 1606-1619. Schuck, P., and Rossmanith, P. 2000. Determination of the sedimentation coefficient distribution by least-squares boundary modeling. Biopolymers 54: 328-341. Schulman, B.A., Kim, P.S., Dobson, C.M., and Redfield, C. 1997. A residue-specific NMR view of the non-cooperative unfolding of a molten globule. Nat. Struct. Biol. 4: 630-634. Schulman, B.A., Redfield, C., Peng, Z.Y., Dobson, C.M., and Kim, P.S. 1995. Different subdomains are most protected from hydrogen exchange in the molten globule and native states of human alpha-lactalbumin. J Mol Biol 253: 651-657. Schwarzinger, S., Kroon, G.J., Foss, T.R., Chung, J., Wright, P.E., and Dyson, H.J. 2001. Sequence-dependent correction of random coil NMR chemical shifts. J Am Chem Soc 123: 2970-2978. Schwarzinger, S., Kroon, G.J., Foss, T.R., Wright, P.E., and Dyson, H.J. 2000. Random coil chemical shifts in acidic M urea: implementation of random coil shift data in NMRView. J Biomol NMR 18: 43-48. Schwieters, C.D., Kuszewski, J.J., Tjandra, N., and Clore, G.M. 2003. The Xplor-NIH NMR molecular structure determination package. J Magn Reson 160: 65-73. Shortle, D. 1993. Denatured states of proteins and their roles in folding and stability. Curr Opin Struct Biol 3: 66-74. Shortle, D., and Abeygunawardana, C. 1993. NMR analysis of the residual structure in the denatured state of an unusual mutant of staphylococcal nuclease. Structure 1: 121-134. Slupsky, C.M., Gentile, L.N., Donaldson, L.W., Mackereth, C.D., Seidel, J.J., Graves, B.J., and McIntosh, L.P. 1998. Structure of the Ets-1 pointed domain and mitogen-activated protein kinase phosphorylation site. Proc Natl Acad Sci USA 95: 12129-12134. Smalla, M., Schmieder, P., Kelly, M., Ter Laak, A., Krause, G., Ball, L., Wahl, M., 162 Bork, P., and Oschkinat, H. 1999. Solution structure of the receptor tyrosine kinase EphB2 SAM domain and identification of two distinct homotypic interaction sites. Protein Sci 8: 1954-1961. Smith, L.J., Bolin, K.A., Schwalbe, H., MacArthur, M.W., Thornton, J.M., and Dobson, C.M. 1996. Analysis of main chain torsion angles in proteins: prediction of NMR coupling constants for native and random coil conformations. J Mol Biol 255: 494-506. Song, J., Jamin, N., Gilquin, B., Vita, C., and Menez, A. 1999. A gradual disruption of tight side-chain packing: 2D 1H-NMR characterization of acid-induced unfolding of CHABII. Nat Struct Biol 6: 129-134. Stapleton, D., Balan, I., Pawson, T., and Sicheri, F. 1999. The crystal structure of an Eph receptor SAM domain reveals a mechanism for modular dimerization. Nat Struct Biol 6: 44-49. Stein, E., Cerretti, D.P., and Daniel, T.O. 1996. Ligand activation of ELK receptor tyrosine kinase promotes its association with Grb10 and Grb2 in vascular endothelial cells. J Biol Chem 271: 23588-23593. Stein, E., Lane, A.A., Cerretti, D.P., Schoecklmann, H.O., Schroff, A.D., Van Etten, R.L., and Daniel, T.O. 1998. Eph receptors discriminate specific ligand oligomers to determine alternative signaling complexes, attachment, and assembly responses. Genes Dev 12: 667-678. Tanford, C. 1968. Protein denaturation. Adv. Protein Chem. 23: 121-282. Tanford, C. 1970. Protein denaturation. C. Theoretical models for the mechanism of denaturation. Adv Protein Chem 24: 1-95. Testoni, B., and Mantovani, R. 2006. Mechanisms of transcriptional repression of cell-cycle G(2)/M promoters by p63. Nucleic Acids Res 34: 928-938. Thanos, C.D., Goodwill, K.E., and Bowie, J.U. 1999. Oligomeric structure of the human EphB2 receptor SAM domain. Science 283: 833-836. Thompson, J.D., Higgins, D.G., and Gibson, T.J. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22: 4673-4680. 163 Tran, H.H. et al. Structure of the wild-type TEL-SAM polymer to be published. van Mierlo, C.P., and Steensma, E. 2000. Protein folding and stability investigated by fluorescence, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy: the flavodoxin story. J. Biotechnol. 79: 281-298. van Mierlo, C.P., van den Oever, J.M., and Steensma, E. 2000. Apoflavodoxin (un)folding followed at the residue level by NMR. Protein Sci. 9: 145-157. van Mierlo, C.P., van Dongen, W.M., Vergeldt, F., van Berkel, W.J., and Steensma, E. 1998. The equilibrium unfolding of Azotobacter vinelandii apoflavodoxin II occurs via a relatively stable folding intermediate. Protein Sci. 7: 2331-2344. van Nuland, N.A., Meijberg, W., Warner, J., Forge, V., Scheek, R.M., Robillard, G.T., and Dobson, C.M. 1998. Slow cooperative folding of a small globular protein HPr. Biochemistry 37: 622-637. Wang, W.K., Bycroft, M., Foster, N.W., Buckle, A.M., Fersht, A.R., and Chen, Y.W. 2001. Structure of the C-terminal sterile alpha-motif (SAM) domain of human p73 alpha. Acta Crystallogr, Sect D: Biol Crystallogr 57: 545-551. Wang, Y., and Shortle, D. 1995. The equilibrium folding pathway of staphylococcal nuclease: Identification of the most stable chain-chain interactions by NMR and CD spectroscopy. Biochemistry 34: 15895-15905. Warren, J.R., and Gordon, J.A. 1966. On the refractive indices of aqueous solutions of urea. J. Phys. Chem. 70: 297–300. Wei, G., Schaffner, A.E., Baker, K.M., Mansky, K.C., and Ostrowski, M.C. 2003. Ets-2 interacts with co-repressor BS69 to repress target gene expression. Anticancer Res 23: 2173-2178. Wetlaufer, D.B. 1973. Nucleation, rapid folding, and globular intrachain regions in proteins. Proc Natl Acad Sci USA 70: 697-701. Wijesinha-Bettoni, R., Dobson, C.M., and Redfield, C. 2001. Comparison of the denaturant-induced unfolding of the bovine and human alpha-lactalbumin molten globules. J. Mol. Biol. 312: 261-273. Wilson, P.J., McGlinn, E., Marsh, A., Evans, T., Arnold, J., Wright, K., Biden, K., Young, J., Wainwright, B., Wicking, C., et al. 2000. Sequence variants of DLC1 in colorectal and ovarian tumours. Hum Mutat 15: 156-165. 164 Wishart, D.S., Bigam, C.G., Holm, A., Hodges, R.S., and Sykes, B.D. 1995. 1H, 13C and 15N random coil NMR chemical shifts of the common amino acids. I. Investigations of nearest-neighbor effects. J Biomol NMR 5: 67-81. Wishart, D.S., and Sykes, B.D. 1994a. The 13C chemical-shift index: a simple method for the identification of protein secondary structure using 13C chemical-shift data. J Biomol NMR 4: 171-180. Wishart, D.S., and Sykes, B.D. 1994b. Chemical-shifts as a tool for structure determination. In Nuclear Magnetic Resonance, Pt C, pp. 363-392. Wishart, D.S., Sykes, B.D., and Richards, F.M. 1992. The chemical shift index: a fast and simple method for the assignment of protein secondary structure through NMR spectroscopy. Biochemistry 31: 1647-1651. Wüthrich, K. 1986. NMR of proteins and nucleic acids. John Wiley & Sons, New York. Xu, Y., Lin, Z., Ho, C., and Yang, D. 2005. A general strategy for the assignment of aliphatic side-chain resonances of uniformly 13C,15N-labeled large proteins. J Am Chem Soc 127: 11920-11921. Yamasaki, K., Ogasahara, K., Yutani, K., Oobatake, M., and Kanaya, S. 1995. Folding pathway of Escherichia coli ribonuclease HI: a circular dichroism, fluorescence, and NMR study. Biochemistry 34: 16552-16562. Yuan, B.Z., Miller, M.J., Keck, C.L., Zimonjic, D.B., Thorgeirsson, S.S., and Popescu, N.C. 1998. Cloning, characterization, and chromosomal localization of a gene frequently deleted in human liver cancer (DLC-1) homologous to rat RhoGAP. Cancer Res 58: 2196-2199. Yuan, B.Z., Zhou, X.L., Durkin, M.E., Zimonjic, D.B., Gumundsdottir, K., Eyfjord, J.E., Thorgeirsson, S.S., and Popescu, N.C. 2003. DLC-1 gene inhibits human breast cancer cell growth and in vivo tumorigenicity. Oncogene 22: 445-450. Zeeb, M., Lipps, G., Lille, H., and Balbach, J. 2004. Folding and association of an extremely stable dimeric protein from Sulfolobus islandicus. J. Mol. Biol. 336: 227-240. Zeeb, M., Rosner, H., Zeslawski, W., Canet, D., Holak, T.A., and Balbach, J. 2002. Protein folding and stability of human CDK inhibitor p19(INK4d). J. Mol. Biol. 315: 447-457. 165 Zhang, H., Smolen, G.A., Palmer, R., Christoforou, A., van den Heuvel, S., and Haber, D.A. 2004. SUMO modification is required for in vivo Hox gene regulation by the Caenorhabditis elegans Polycomb group protein SOP-2. Nat Genet 36: 507-511. Zhang, H., Xu, Q.L., Krajewski, S., Krajewska, M., Xie, Z.H., Fuess, S., Kitada, S., Pawlowski, K., Godzik, A., and Reed, J.C. 2000. BAR: An apoptosis regulator at the intersection of caspases and Bcl-2 family proteins. Proc Natl Acad Sci USA 97: 2597-2602. Zhong, D., Zhang, J., Yang, S., Soh, U.J., Buschdorf, J.P., Zhou, Y.T., Yang, D., and Low, B.C. 2009. The SAM domain of the RhoGAP DLC1 binds EF1A1 to regulate cell migration. 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