Molecular mechanisms underlying the thermal stability and acid induced unfolding of CHABII

MOLECULAR MECHANISMS UNDERLYING THE THERMAL STABILITY AND ACID-INDUCED UNFOLDING OF CHABII WEI ZHENG A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF BIOLOGICAL SCIENCES NATIONAL UNIVERSITY OF SINGAPORE 2005 Acknowledgements Firstly, I’d like to extend as many thanks as possible to my supervisor, Dr Song Jianxing During my graduate study in the past two years, he not only provided me a lot of useful instructions on my project, but also generously shared his valuable scientific experience with me He always gave me courage to overcome all the obstacles in my research His patience and consideration also allowed me to work happily and comfortably in the lab everyday I learned a lot from his great enthusiasm for science and life, which, I believe, will benefit me a lot along all my life I also want to express my gratitude to all my labmates They were very warm-hearted and friendly, giving me help whenever I needed They were always open for discussion and shared their stimulating ideas with me I really cherished our valuable friendship and enjoyed all the days spent with them Furthermore, I really appreciate all the support and kindly help from all my friends Their encouragements always gave me energy and I therefore never felt lonely Their experience also enriched my life, and my life became colorful because of them Especially, I want to take this opportunity to give my gratitude to my parents, who gave all of their love to me and shared all my happiness and sadness, successes and failures during the past 24 years Finally, I am grateful to National University of Singapore for providing me a research scholarship, which guaranteed my research and living in I Singapore Living and studying here has been a wonderful experience in my life II Contents Acknowledgements I Summary VI List of Table(s) VIII List of Figures IX List of Symbols and Abbreviations XI Publication List XIII CHAPTER I Introduction Part I Protein Folding and Stability 1.1.1 Overview of Protein Folding Theories 1 1.1.1.1 Framework Model 1.1.1.2 Hydrophobic Collapse Model 1.1.1.3 Nucleation-Condensation Model 1.1.1.4 Energy Landscape Theory 1.1.2 Molten Globules 1.1.2.1 Structural Characteristics of Molten Globules 1.1.2.1.1 Compactness 1.1.2.1.2 Secondary Structure and Native-like Tertiary Fold 1.1.2.1.3 Dynamics and Hydration 1.1.2.2 Kinetic Role of Molten Globules in Folding 1.1.2.2.1 Equilibirium Intermediates=Kinetic Intermediates 1.1.2.2.2 On-pathway or Off-Pathway Intermediates 1.1.3 Cooperativity of Protein Unfolding 17 1.1.4 Protein Stability 20 1.1.4.1 Van der Waal interactions 1.1.4.2 Hydrophobic interactions 1.1.4.3 Hydrogen Bonds III 1.1.4.4 Electrostatic Interactions 1.1.4.5 Conformational Entropy 1.1.4.6 Disulfide Bonds 1.1.5 Previous Studies about CHABII Part II Fundamentals of NMR 24 26 1.2.1 Nuclear Spins and NMR Signals 26 1.2.2 Several NMR Parameters 27 1.2.2.1 Chemical Shift 1.2.2.2 Coupling Constant 1.2.2.3 Nuclear Overhauser Enhancement (NOE) 1.2.3 Structure Calculation 30 1.2.4 Chemical Exchanges Measured by NMR 30 Part III Objectives 32 CHAPTER II Material and Methods 2.1 Gene Syntheses of CHABII and [Phe21]-CHABII 34 2.2 Expression Vector Construction 35 2.3 Preparation of Competent E.coli Cells 36 2.4 Transformation of E.coli Cells 36 2.5 Protein Expression and Purification 37 2.6 Determination of Protein Concentration by Spectroscopy 39 2.7 Refolding of CHABII and [Phe21]-CHABII In vitro 39 2.8 CD Characterization of pH-induced and Thermal Unfolding 40 2.9 NMR Experiments and Structure Calculations 40 Chapter III Results and Discussion 3.1 Expression Vector Construction 43 3.2 Protein Expression and Purification 46 3.3 Refolding of CHABII and [Phe21]-CHABII in vitro 48 3.4 pH-induced Unfolding Characterized by CD 51 IV 3.5 Thermal Unfolding Characterized by CD 3.6 NMR Resonance Assignments and Three-dimensional Structure Determination 53 56 3.6.1 Sequential Assignments 56 3.6.2 Conformational shifts 57 3.6.3 NOE Patterns and Assignments 59 3.6.4 3D Structure Determination 65 3.7 pH-induced unfolding monitored by HSQC spectroscopy 69 3.8 Discussion 75 References 81 Appendices 89 Proton Chemical Shifts of CHABII at pH 6.3 and 293 K Proton Chemical Shifts of CHABII at pH 4.0 and 293 K Proton Chemical Shifts of [Phe21]-CHABII at pH 6.3 and 293 K Proton Chemical Shifts of [Phe21]-CHABII at pH 4.0 and 293 K V Summary The 37-residue protein CHABII was previously demonstrated to undergo a gradual pH-induced unfolding It has been shown that even at pH 4.0 CHABII still retained a highly native-like secondary structure and tertiary topology although its tight side-chain packing was severely-disrupted, typical of the molten globule state In this regard, CHABII at pH 4.0 may offer an attractive model for deeper understanding of the molten globule state or partially-folded proteins In the present study, we expressed and refolded the recombinant proteins of CHABII and its mutant [Phe21]-CHABII, and subsequently conducted extensive CD and NMR characterizations The results indicated: 1) Replacement of His21 by Phe in [Phe21]-CHABII eliminated the pH-induced unfolding from pH 6.5 to 4.0, indicating the critical role of His21 in the pH-induced unfolding of CHABII Further examination revealed that although the pH-induced unfolding of CHABII was also triggered by the protonation of His residue as previously observed for apomyoglobin, their molecular mechanisms are very different 2) Replacement of His21 by Phe with higher side chain hydrophobicity caused no significant structural rearrangement but considerably enhanced the packing interaction of the hydrophobic core, as evident from a dramatic increase in NOE contacts in [Phe21]-CHABII The enhancement led to an increase of the thermal stability of [Phe21]-CHABII by ~17 degree This observation highlights the complexity of determinants of protein thermal stability and further implies the limitation to rationalize protein VI stability only based on the three-dimensional structure knowledge 3) Monitoring the pH-induced unfolding by 1H-15N HSQC spectroscopy allowed to visualize the gradual development of the CHABII molten globule At pH 4.0, the HSQC spectrum of CHABII was poorly-dispersed with dispersions of ~1 ppm over proton dimension and 10 ppm over 15 N dimension, characteristic of severely or even “completely unfolded” proteins On the other hand, unambiguous assignments of persistent NOEs, in particular medium- and long-range NOEs defining tertiary packing, indicated that CHABII at pH 4.0 also had a highly native-like topology This strongly implies that the degree of the native-like topology might be significantly underestimated in the previous characterization of partially-folded and even “completely-unfolded” proteins VII List of Table(s) Table NMR restraints used for structure calculation and structural statistics for the 10 selected lowest-energy structures VIII List of Figures Figure pGEX-4T-1-CHABII and pGEX-4T-1-[Phe21]-CHABII vector constructions (a) PCR-based gene syntheses; (b) PCR screenings for positive colonies; (c) Double-digestion identifications for positive colonies Figure Automated DNA sequencing results for pGEX-4T-1-CHABII and pGEX-4T-1-[Phe21]-CHABII, (a) and (b) respectively Figure CHABII Expression and Purification Monitored by SDS-PAGE (a) Expression and purification of CHABII-GST fusion protein; (b) Thrombin digestion of CHABII-GST fusion protein Figure CHABII immediately released from in-gel thrombin-cleavage of GST-CHABII fusion protein (a) The analytic HPLC profile on an RP-18 column; (b) and (c) The far-UV CD spectra of the 1st and 2nd peak appearing in the HPLC profile recorded at 20 ℃, pH 6.8, respectively Figure Disulfide-related refolding of CHABII monitored by HPLC on an analytic RP-18 column (a) The HPLC profile of misfolded CHABII species before refolding (b) The HPLC profile of CHABII after refolding for three hours in the redox buffer containing reduced and oxidized glutathione Figure pH-induced unfolding monitored by far-UV circular dichroism (CD) spectroscopy (a) Far-UV CD spectra of [Phe21]-CHABII at pH 6.5, 5.5, 4.6 and 4.0 (b) Far-UV CD spectra of CHABII at pH 6.5, 5.5, 4.6 and 4.0 Figure Thermal unfolding of CHABII and [Phe21]-CHABII at different pH values with a temperature range from 20 to 95 ºC followed by monitoring changes in ellipticity at 220 nm (a) Thermal unfolding curves of CHABII and [Phe21]-CHABII at pH 6.5 (b) Thermal unfolding curves of CHABII and [Phe21]-CHABII at pH 4.0 (c) Thermal unfolding curves of [Phe21]-CHABII at pH 4.0 and [Phe21]-CHABII at pH 6.5 (d) Thermal unfolding curves of CHABII at pH 4.0 and CHABII at pH 6.5 Figure NOESY spectrum of [Phe21]-CHABII (500 MHz, mixing time of 250 ms) acquired at pH 4.0 and 20 ℃ Figure CαH conformational shifts of CHABII and [Phe21]-CHABII at 20 ºC (a) CαH conformational shifts of [Phe21]-CHABII at pH 6.5 and pH 4.0; (b) CαH conformational shifts of CHABII at pH 6.5 and pH 4.0 Figure 10 NOE constraints of CHABII and [Phe21]-CHABII at 20 ºC (a) NOE constraints of [Phe21]-CHABII at pH 6.5 (b) NOE constraints of IX Brunger, A.T., Clore, G.M., Gronenborn, A.M., Saffrich, R., and Nilges, M (1993) Assessing the quality of solution nuclear magnetic resonance structures by complete cross-validation Science 261, 328-31 Brunger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.-S., Kuszewski, J., Nilges, M., Pannu, N.S., Read, R.J., Rice, L.M., Simonson, T and Warren, G.L (1998).Crystallography & NMR system (CNS): a new software system for macromolecular structure determination Acta Crystallogr D54: 905–921 Bu, Z., Neumann, D.A., Lee, S.H., Brown, C.M., Engelman, D.M and Han, C.C (2002) A view of dynamics changes in the molten globule-native folding step by quasielastic neutron scattering J Mol Biol 301, 525-36 Cornilescu G, Delaglio F, Bax A (1999) Protein backbone angle restraints from searching a database for chemical shift and sequence homology J Biomol NMR 13, 289-302 Denisov, V.P., Jonsson, B.H and Halle B (1999) Hydration of denatured and molten globule proteins Nature Struct Biol 6, 253-260 Dill, K.A., Bromberg, S., Yue, K., Fiebig, K.M., Yee, D.P., Thomas, P.D and Chan, H.S (1995) Principles of protein folding a perspective from simple exact models Protein Science 4, 61-602 Dill K.A and Chan H.S., (1997) From Levinthal to pathways to funnels Nature Struct Biol 4, 10-19 Dobson, CM., and Karplus, M (1999) The fundamentals of protein folding: Bringing together theory and experiment Curr Opin Struct Biol 9, 92-101 Dolgikh, D.A., Gilmanshin, R I., Brazhnikov, E.V., Bychkova, V.E., Semisotnov, G.V., Venyaminov, S.Yu., and Ptitsyn, O.B (1981) Alpha-Lactalbumin: compact state with fluctuating tertiary structure? FEBS Lett 136, 311-315 Drakopoulou, E., Vizzavona, J., Neyton, J., Aniort V., Bouet, F., Virelizier, H., Menez, A and Vita, C (1998) Consequence of the removal of evolutionary conserved disulfide bridges on the structure and function of charybdotoxin and evidence that particular cysteine spacings govern specific disulfide bond formation Biochemistry 37, 1292-1301 Dyson, H.J and Wright, P.E (2002) Insights into the structure and dynamics of unfolded proteins from nuclear magnetic resonance Adv Protein Chem 62, 311-40 82 Dyson, H.J and Wright, P.E (2004) Unfolded proteins and protein folding studied by NMR Chem Rev 104, 3607-22 Elove,G.A., Bhuyan, A.K., and Roder, H (1994) Kinetic mechanism of cytochrome c folding: involvement of the heme and its ligands Biochemistry 33, 6925-6935 Balbach,J., Forge,V., Van Nuland, N.A.J., Winder, S.L., Hore, P.L., and Dobson, C.M (1995) Following protein folding in real time using NMR spectroscopy Nature Struct Biol 2, 865-870 Englander, S.W., and Mayne, L.C (1992) Protein folding studied using hydrogen-exchange labeling and two-dimensional NMR Annu Rev Biophys Biomol Struct 21, 243-265 Feng, H., Vu, N-D., Zhou Z., Bai, Y.W (2004) Structural examination of Phi-value analysis in protein folding Biochemistry 43, 14325-14331 Fersht, A.R (1997) Nucleation mechanisms in protein folding Curr Opin Struct Biol 7, 3-9 Fersht, A.R and Daggett, V (2002) Protein folding and unfolding at atomic resolution Cell 108, 573-582 Friebolin, H (1998) Basic One- and Two- dimentional NMR Spectroscopy Weinheim: New York: Wiley-VCH, 3rd rev ed Garcia, C., Nishimura, C., Cavagnero, S., Dyson, H.J and Wright, P.E (2002) Changes in the apomyoglobin folding pathway caused by mutation of the distal histidine residue Biochemistry 39, 11227-37 Geierstanger, B., Jamin, M., Volkman, B.F and Baldwin, R.L (1998) Protonation behavior of histidine 24 and histidine 119 in forming the pH folding intermediate of apomyoglobin Biochemistry 37, 4254-65 Griko,Y.V., Freire, E and Privalov, P.L (1994) Energetics of the alpha-lactalbumin states: a calorimetric and statistical thermodynamic study Biochemistry 33, 1889-1899 Heidary, D.K., O’Neill, J.C.Jr., Roy, M and Jennings, P.A (2000) An essential intermediate in the folding of dihydrofolate reductase Proc Natl Acad Sci USA 97, 5866-5870 83 Hughson, F.M., Wright, P.E., and Baldwin, R.L (1990) Structural characterization of a partly folded apomyoglobin intermediate Science 249, 1544-1548 Ikeguchi, M., Kuwajima, K., Mitani M., and Sugai, S (1986) Evidence for identity between the equilibrium unfolding intermediate and a transient folding intermediate: a comparative study of the folding reactions of alpha-lactalbumin and lysozyme Biochemistry 25, 6965-6972 Jaenicke, R and Bohm, G (1998) The stability of proteins in extreme environments Curr Opin Struct Biol 8, 738-48 Jeener, J., Meier, B.H., Bachmann, P and Ernst, R.R (1979) Investigation of exchange processes by two-dimensional NMR spectroscopy J Chem Phys 71, 4546-4554 Jeng, M.F., Englander, S.W., Elove, G.A., Wang, A.I., and Roder, H (1990) Structural description of acid-denatured cytochrome c by hydrogen exchange and 2D NMR Biochemistry 29, 10433-10437 Jennings, P.A., and Wright, P.E (1993) Formation of a molten globule intermediate early in the kinetic folding pathway of apomyoglobin Science 262, 892-896 Johnson, B.A and Blevins, R.A (1994) NMRView: A computer program for the visualization and analysis of NMR data J Biomol NMR 4, 603-614 Kamagata, K., Arai, M., and Kuwajima, K (2004) Unification of the folding mechanisms of non-two-state and two-state proteins J Mol Biol 339, 951-965 Kataoka, M., Kuwajima, K., Tokunaga, F., and Goto, Y (1997) Structural characterization of molten globule of α-lactalbumin by solution X-ray scattering Protein Sci 6, 422-430 Kay, M.S and Baldwin, R.L (1996) Packing interactions in the apomyglobin folding intermediate Nature Struct Biol 3, 439-45 Khorasanizadeh, S., Peters, I.D and Roder, H (1996) Evidence for a three-state model of protein folding from kinetic analysis of ubiquitin variants with altered core residues Nature Struct Biol.3 193-205 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 84 Biochem 51, 459-489 Kim, P.S and Baldwin, R.L (1990) Intermediates in the folding reactions of small proteins Annu Rev Biochem 59, 631-60 Klein-Seetharaman J, Oikawa M, Grimshaw SB, Wirmer J, Duchardt E, Ueda T, Imoto T, Smith LJ, Dobson CM, Schwalbe H (2002) Long-range interactions within a nonnative protein Science 295, 1719-1722 Koradi, R., Billeter, M and Wuthrich, K (1996) MOLMOL: a program for display and analysis of macromolecular structures J Mol Graphics 14, 51-55 Kuwajima, K., Nitta, K., Yoneyama, M., and Sugai, S (1976) Three-state denaturation of -lactalbumin by guanidine hydrochloride J Mol Biol 106, 359-373 Kuwajima, K., Hiraoka,Y., Ikeguchi, M., and Sugai, S (1985) Comparison of the transient folding intermediates in lysozyme and alpha-lactalbumin Biochemistry 24, 874-881 Kuwajima, K (1989) The molten globule state as a clue for understanding the folding and cooperativity of globular-protein structure Proteins 6, 87-103 Lakshmikanth, G.S., Sridevi, K., Krishnamoorthy, G and Udgaonkar, J.B (2001) Structure is lost incrementally during the unfolding of barstar Nature Struct Biol 8, 799-804 Laurents, D.V., Bruix, M., Jamin, M and Baldwin, R.L (1998) A pulse-chase-competition experiment to determine if a folding intermediate is on or off-pathway: application to ribonuclease A J Mol Biol 283, 669-678 Levinthal C (1968) Are there pathways for protein folding? J Chem Phys 65, 44-45 Loh, S.N., Kay, M.S., and Baldwin, R.L.(1995) Structure and stability of a second molten globule intermediate in the apomyoglobin folding pathway Proc Natl Acad Sci USA 92, 5446-5450 Maity H., Maity M and Englander S.W (2004) How cytochrome c folds, and why: submolecular foldon units and their stepwise sequential stabilization J Mol Biol 343, 223-33 Matouschek, A., Kellis, J.T.Jr., Serrano, L., Bycroft, M., and Fersht, A.R (1990) Transient folding intermediates characterized by protein engineering Nature 346, 85 440-445 Murphy, K.P (1995) Chapter 1, Protein folding and Stability: Theory and Practice Edited by Shirley B.A Nosoh, Y and Sekiguchi, T (1991) Protein Stability and Stabilization Through Protein Engineering Ohgushi, M., and Wada, A (1983) "Molten-globule state": A compact form of globular proteins with mobile side-chains FEBS Lett 164, 21-24 Pace, C.N., Vajdos, F., Fee, L., Grimsley, G., and Gray, T (1995) How to measure and predict the molar absorption coefficient of a protein Protein Science 4, 2411-2423 Peng, Z., and Kim, P.S (1994) A protein dissection study of a molten globule Biochemistry 33, 2136-2141 Perutz, M.F (1978) Electrostatic effects in proteins Science 201, 1187-1191 Privalov, P.L (1979) Stability of Proteins: Small Globular Proteins Adv Protein Chem 33, 167-241 Ptitsyn, O.B (1994) Kinetic and equilibrium intermediates in protein folding Prot Eng 7, 593-596 Ptitsyn, O.B (1995a) Molten globule and protein folding Adv Pro Chem 47, 83-229 Ptitsyn, O.B (1995b) Structure of folding intermediates Curr Opin Struct Biol 5, 74-78 Radford, S.E., Dobson, C.M and Evans, P.A (1992) The folding of hen lysozyme involves partially structured intermediates and multiple pathways Nature 358, 302-307 Radford, S.E., Woolfson, D.N., Martin, S.R., Lowe, G and Dobson, C.M (1991) A three-disulphide derivative of hen lysozyme Structure, dynamics and stability Biochem J 273(Pt 1), 211-7 Raschke, T.M., and Marqusee, S (1997) The kinetic folding intermediate of ribonuclease H resembles the acid molten globule and partially unfolded molecules detected under native conditions Nature Struct Biol 4, 298-304 86 Redfield, C (2004) NMR studies of partially folded molten-globule states Methods Mol Biol 278, 233-54 Sadqi, M., Lapidus, L.J and Munoz, V (2003) How fast is protein hydrophobic collapse? Proc Natl Acad Sci USA 100, 12117-12122 Sattler, M., Schleucher, J and Griesinger, C (1999) Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients Prog NMR Spectrosc 34, 93-158 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, 651-7 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 Nature Struct Biol 4, 630-634 Semisotnov, G.V., Kutyshenko, V.P., and Ptitsyn, O.B (1989) Intramolecular mobility of a protein in a "molten globule" state.A study of carbonic anhydrase by 1H-NMR Mol Biol (Moscow) 23, 808-815 Shi J, Wei Z and Song J (2004) Dissection study on the severe acute respiratory syndrome 3C-like protease reveals the critical role of the extra domain in dimerization of the enzyme: defining the extra domain as a new target for design of highly specific protease inhibitors J Biol Chem 279, 24765-24773 Shortle, D and Ackerman, M.S (2001) Persistence of native-like topology in a denatured protein in M urea Science 293, 487-9 Song, J., Bai, P., Luo, L and Peng, Z.Y (1998) Contribution of individual residues to formation of the native-like tertiary topology in the alpha-lactalbumin molten globule J Mol Biol 280, 167-74 Song, J., Gilquin, B., Jamin, N., Drakopoulou, E., Guenneugues M., Dauplais, M., Vita, C and Menez, A (1997) NMR solution structure of a two-disulfide derivative of charybdotoxin: structural evidence for conservation of scorpion toxin alpha/beta motif and its hydrophobic side chain packing Biochemstry 36, 3760-3766 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 Nature Struct Biol 6, 129-134 87 Uversky, V.N (1993) Use of fast protein size-exclusion liquid chromatography to study the unfolding of proteins which denature through the molten globule Biochemistry 32, 13288-13298 Uversky, V N., and Ptitsyn, O.B (1994) "Partly folded" state - a new equilibrium state of protein molecules Four-state GdmCl-induced unfolding of beta-lactamase at low temperature Biochemistry 33, 2782-2791 Vieille, C and Zeikus, G.J (2001) Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability Microbiol Mol Biol Rev 65, 1-43 Wagner, G and Wuthrich, K (1982) Sequential resonance assignments in protein H nuclear magnetic resonance spectra Basic pancreatic trypsin inhibitor J Mol Biol 155, 347-66 Wang, F., and Tang, X J (1996) Conformational heterogeneity and stability of Apomyoglobin studied by hydrogen/deuterium and electrospray ionization mass spectrometry Biochemistry 35, 4069-4078 Wright, C.F., Lindorff-Larsen, K., Randles, L.G., Clarke, J (2003) Parallel protein-unfolding pathways revealed and mapped Nature Struct Biol 10, 658-662 Wu, L.C and Kim, P.S (1998) A specific hydrophobic core in the alpha-lactalbumin molten globule J Mol Biol 280, 175-182 Wu, L.C., Peng Z., and Kim P.S (1995) Bipartite structure of the alpha-lactalbumin molten globule Nature Struct Biol 2, 281-285 Wuthrich, K (1986) NMR of Proteins and Nucleic Acids, John Wiley, New York Wuthrich, K (2003) NMR studies of structure and function of biological macromolecules (Nobel lecture) Angew Chem Int Ed 42, 3340-3363 88 Appendix I Proton Chemical Shifts of CHABII at pH 6.3 and 293 K Gln1 NH 8.600 Hα 4.386 Hβ 1.619 1.485 3.172 2.991 4.268 2.959 2.747 2.037 Phe2 8.784 4.646 Thr3 Asn4 7.901 8.612 4.654 4.835 Val5 8.365 4.071 Ser6 8.530 4.946 Cys7 7.970 4.780 Thr8 Thr9 Ser10 9.416 7.823 4.512 4.898 Lys11 Glu12 Ala13 Trp14 7.630 7.485 8.628 3.547 4.082 3.905 4.110 3.937 3.842 3.275 2.758 4.260 4.575 4.062 3.939 1.648 2.117 1.344 3.455 Ser15 Val16 7.731 7.888 4.102 3.637 4.008 2.099 Cys17 8.499 4.339 Gln18 8.290 3.866 2.999 2.841 1.908 Arg19 7.485 4.094 1.895 Leu20 8.217 4.165 1.596 His21 7.931 3.283 Hγ 1.887 others δNH2: 7.277 δNH2: 6.653 2,6H: 7.221 3,5H: 7.292 1.178 δNH2: 7.624 δNH2: 6.935 0.995 0.921 1.202 1.206 1.345 2.277 1NH: 9.972 2H: 7.068 4H: 7.634 5H: 7.115 6H: 7.217 7H: 7.497 1.016 0.885 2.085 1.683 1.612 1.016 δNH2: 7.091 δNH2: 6.646 δCH2: 3.184 εNH: 7.225 δCH3: 0.850 δCH3: 0.764 4H: 6.822 89 Asn22 8.069 4.646 Thr23 Ser24 Arg25 7.548 8.295 8.021 4.641 4.536 4.244 Gly26 7.800 Lys27 9.094 5.081 3.771 4.662 Cys28 9.105 4.835 Met29 8.730 4.774 4.333 Asn30 Lys31 8.628 3.992 Lys32 7.657 5.140 Ala33 Arg34 8.452 9.266 4.509 4.891 Cys35 8.557 5.481 Tyr36 8.401 4.898 Ser37 8.252 4.291 2.866 3.038 2.710 4.205 3.795 1.643 1.848 1.735 2.866 2.510 2.034 1.723 2.984 2.752 2.247 2.103 1.714 1.470 1.210 1.730 1.596 3.125 2.368 3.077 2.686 3.845 3.771 2H: 7.824 δNH2: 7.601 δNH2: 6.846 1.079 1.483 1.372 1.400 2.384 1.356 1.273 1.333 2,6H: 6.979 3,5H: 6.584 90 Appendix II Proton Chemical Shifts of CHABII at pH 4.0 and 293 K Gln1 NH 8.634 Hα 4.398 Hβ 1.811 1.726 3.168 2.997 4.230 2.944 2.746 2.095 Hγ 2.116 Phe2 8.691 4.686 Thr3 Asn4 7.958 8.557 4.558 4.860 Val5 8.364 4.135 Ser6 8.476 4.905 3.934 3.853 Cys7 Thr8 Thr9 Ser10 Lys11 8.011 9.192 7.778 4.492 4.905 4.285 1.219 8.134 3.849 1.733 1.633 1.391 Glu12 Ala13 Trp14 7.719 7.574 8.661 4.115 3.929 4.082 1.361 3.457 Ser15 Val16 7.892 7.832 4.071 3.644 3.994 2.115 Cys17 8.315 4.286 Gln18 8.312 3.937 2.964 2.751 1.851 Arg19 7.440 4.079 1.875 Leu20 7.838 4.193 His21 8.135 4.748 1.700 1.608 3.337 3.089 others δNH2: 7.407 δNH2: 6.738 2,6H: 7.236 3,5H: 7.313 1.130 δNH2: 7.626 δNH2: 6.943 0.974 0.914 2.336 1NH: 10.049 2H: 7.186 4H: 7.622 5H: 7.090 6H: 7.201 7H: 7.485 1.012 0.875 2.041 1.715 1.607 1.200 δNH2: 7.052 δNH2: 6.712 δCH2: 3.179 εNH: 7.189 δCH3: 0.846 δCH3: 0.774 4H: 7.130 91 Asn22 8.526 4.664 Thr23 Ser24 7.755 8.315 4.499 4.526 Arg25 8.175 4.177 Lys27 9.003 5.165 3.644 4.642 Cys28 9.080 4.883 Met29 8.747 4.758 Asn30 9.558 4.350 Lys31 8.599 4.028 Lys32 7.685 5.106 Ala33 Arg34 8.531 9.242 4.481 4.886 Cys35 8.589 5.531 Tyr36 8.550 4.865 Ser37 8.256 4.310 Gly26 2.953 2.787 4.293 3.809 3.774 1.700 1.851 1.743 2.885 2.534 2.043 1.754 2.980 2.745 2.237 2.096 1.724 1.474 1.224 1.733 1.650 3.199 2.504 3.096 2.749 3.805 δNH2: 7.606 δNH2: 6.902 1.127 1.603 1.441 δCH2: 3.037 εNH: 7.049 1.398 2.397 1.378 1.357 δCH2: 2.912 εNH: 6.931 2,6H: 7.008 3,5H: 6.592 92 Appendix III Proton Chemical Shifts of [Phe21]-CHABII at pH 6.3 and 293 K Gln1 NH 8.624 Hα 4.373 Hβ 1.607 1.361 3.159 2.990 4.276 2.977 2.751 2.036 Phe2 8.774 4.635 Thr3 Asn4 7.823 8.686 4.674 4.924 Val5 8.420 4.039 Ser6 8.565 4.945 Cys7 7.978 4.766 Thr8 Thr9 Ser10 9.406 7.782 4.518 4.874 Lys11 Glu12 7.986 7.637 3.524 4.072 3.934 3.843 3.249 2.772 4.270 4.579 4.066 3.924 1.636 2.146 Ala13 Trp14 7.507 8.677 3.900 4.077 1.355 3.457 Ser15 Val16 7.692 7.978 4.144 3.657 4.048 2.153 Cys17 8.622 4.510 Gln18 8.293 3.962 3.077 2.863 1.974 Arg19 7.758 4.077 1.881 Leu20 8.411 4.074 1.546 1.402 Hγ 1.787 others δNH2: 7.111 δNH2: 6.592 2,6H: 7.220 3,5H: 7.283 1.081 δNH2: 7.633 δNH2: 6.937 1.036 0.971 1.215 1.208 1.347 2.290 2.236 1NH: 9.936 2H: 7.034 4H: 7.685 5H: 7.144 6H: 7.238 7H: 7.519 1.050 0.924 2.168 1.695 1.594 0.786 δNH2: 7.166 δNH2: 6.640 δCH2: 3.187 εNH: 7.224 δCH3: 0.679 δCH3: 0.474 93 Phe21 7.802 4.871 3.472 2.821 Asn22 7.922 4.691 Thr23 Ser24 Arg25 7.478 8.236 7.797 4.709 4.517 4.225 3.164 2.707 4.163 3.836 1.610 Gly26 7.715 Lys27 9.094 4.931 3.922 4.679 Cys28 9.083 4.863 Met29 8.756 4.777 4.320 Asn30 Lys31 8.633 3.990 Lys32 7.653 5.157 Ala33 Arg34 8.475 9.262 4.511 4.934 Cys35 8.463 5.439 Tyr36 8.292 4.889 Ser37 8.194 4.281 1.856 1.735 2.862 2.517 2.034 1.714 2.980 2.762 2.258 2.100 1.735 1.471 1.197 1.733 1.577 3.175 2.195 3.065 2.671 3.838 3.725 3,5H: 7.145 2,6H: 7.302 4H: 7.222 δNH2: 7.590 δNH2: 6.826 1.092 1.445 1.384 δCH2: 3.045 εNH: 7.037 1.451 1.384 2.369 1.372 1.268 1.290 1.210 δCH2: 2.788 εNH: 6.944 2,6H: 6.944 3,5H: 6.572 94 Appendix IV Proton Chemical Shifts of [Phe21]-CHABII at pH 4.0 and 293 K Gln1 NH 8.591 Hα 4.357 Hβ 1.583 1.326 3.161 2.994 4.246 2.974 2.748 2.039 Phe2 8.765 4.657 Thr3 Asn4 7.827 8.696 4.684 4.933 Val5 8.427 4.045 Ser6 8.559 4.941 Cys7 7.980 4.778 Thr8 Thr9 Ser10 9.288 7.754 9.199 4.520 4.874 4.828 Lys11 Glu12 7.957 7.626 3.523 4.073 3.942 3.839 3.250 2.770 4.269 4.580 4.061 3.912 1.639 2.146 Ala13 Trp14 7.482 8.640 3.903 4.079 1.345 3.461 Ser15 Val16 7.718 7.981 4.145 3.659 4.053 2.148 Cys17 8.596 4.508 Gln18 8.295 3.950 3.073 2.875 1.974 Arg19 7.758 4.078 1.896 Leu20 8.410 4.069 1.548 1.398 Hγ 1.748 others δNH2: 7.090 δNH2: 6.594 2,6H: 7.224 3,5H: 7.276 1.087 δNH2: 7.633 δNH2: 6.934 1.031 0.970 1.203 1.205 1.344 2.345 2.289 1NH: 9.926 2H: 7.035 4H: 7.686 5H: 7.146 6H: 7.233 7H: 7.509 1.048 0.922 2.167 1.670 1.586 0.786 δNH2: 7.170 δNH2: 6.642 δCH2: 3.199 εNH: 7.224 δCH3: 0.674 δCH3: 0.446 95 Phe21 7.799 4.882 3.465 2.820 Asn22 7.919 4.701 Thr23 Ser24 Arg25 7.479 8.232 7.821 4.719 4.518 4.239 3.162 2.710 4.149 3.831 1.641 Gly26 7.727 Lys27 9.093 4.929 3.936 4.681 Cys28 9.029 4.883 Met29 8.757 4.762 Asn30 9.553 4.322 Lys31 8.633 3.990 Lys32 7.653 5.155 Ala33 Arg34 8.465 9.250 4.512 4.933 Cys35 8.476 5.431 Tyr36 8.266 4.899 Ser37 8.199 4.282 1.856 1.739 2.842 2.516 2.040 1.713 2.978 2.763 2.255 2.100 1.731 1.482 1.194 1.723 1.589 3.147 2.209 3.061 2.674 3.834 3.714 3,5H: 7.145 2,6H: 7.299 4H: 7.221 δNH2: 7.591 δNH2: 6.830 1.089 1.470 1.393 δCH2: 3.048 εNH: 7.031 1.451 1.405 2.375 1.356 1.268 1.316 1.245 δCH2: 2.848 δCH2: 2.784 εNH: 6.954 2,6H: 6.954 3,5H: 6.559 96 ... curves of CHABII and [Phe21] -CHABII at pH 6.5 (b) Thermal unfolding curves of CHABII and [Phe21] -CHABII at pH 4.0 (c) Thermal unfolding curves of [Phe21] -CHABII at pH 4.0 and [Phe21] -CHABII at... proteins Extensive 32 CD and NMR experiments were performed to characterize the role of His21 in the pH -induced unfolding of CHABII Thermal unfoldings of CHABII and [Phe21] -CHABII were monitored... Wei Z and Song J (2005) Molecular mechanism underlying the thermal stability and pH -induced unfolding of CHABII J Mol Biol 348 (1):205-18 Shi J., Wei Z and Song J (2004) Dissection study on the