Abstract
Little is known about the kinetic process in which stable intermediates in protein folding are formed: whether their folding is highly cooperative (two-state) or weakly cooperative is controversial. We report here that the folding and unfolding kinetics of the pH 4-stable intermediate (I1) of apomyoglobin are measurable, in the millisecond time range, when monitored by stopped-flow measurements of tryptophan fluorescence. The kinetics confirm that folding of I1 is strongly cooperative, but there is a burst phase (missing amplitude) in unfolding. If the faster steps in unfolding of I1 can be measured directly by suitable fast-reaction methods, they will give information about the nature of the folding transition.
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References
Tanford, C. Protein denaturation. Adv. Prot. Chem. 23, 122–282 (1968).
Privalov, P.L. Stability of proteins: small globular proteins. Adv. Prot. Chem. 33, 167–241 (1979).
Kim, P.S. & Baldwin, R.L. Intermediates in the folding reactions of small proteins. Ann. Rev. Biochem. 59, 631–660 (1990).
Kuwajima, K., Yamaya, H., Miwa, S., Sugai, S. & Nagamura, T. Rapid formation of secondary structure framework in protein folding studied by stopped-flow circular dichroism. FEBS Lett. 221, 115–118 (1987).
Baldwin, R.L. Pulsed H/D exchange studies of folding intermediates. Curr. Opin. Struct. Biol. 3, 84–91 (1993).
Wolynes, P.G., Onuchic, J.N. & Thirumalai, D. Navigating the folding routes. Science 267, 1619–1620 (1995).
Ikeguchi, M., Kuwajima, K., Mitani, M. & Sugai, S. Evidence for identity between the equilibrium unfolding intermediate and a transient folding intermediate: a comparative study of the folding reactions of α-lactalbumin and lysozyme. Biochemistry 25, 6965–6972 (1986).
Jennings, P.A. & Wright, P.E. Formation of a molten globule intermediate early in the kinetic folding pathway of apomyoglobin. Science 262, 892–896 (1993).
Griko, Y.V., Privalov, P., Venyaminov, Y.S. & Kutyshenko, V.P. Thermodynamic study of the apomyoglobin structure. J. Mol. Biol. 202, 127–138 (1988).
Hughson, F.M., Wright, P.E. & Baldwin, R.L. Structural characterization of a partly folded apomyoglobin intermediate. Science 249, 1544–1548 (1990).
Kay, M.S. & Baldwin, R.L. Packing in the apomyoglobin folding intermediate. Nature Struct. Biol. 3, 439–445 (1996).
Loh, S.N., Kay, M.S., Baldwin, R.L. Structure and stability of a second molten globule intermediate in the apomyoglobin folding pathway. Proc. Natl. Acad. Sci. USA 92, 5446–5450 (1995).
Roder, H. Structural characterization of protein folding intermediates by proton magnetic resonance and hydrogen exchange. Meth. Enzymol. 176, 446–473 (1989).
Schmid, F.X. Kinetics of unfolding and refolding in single-___domain proteins. in Protein Folding (ed. I.E. Creighton) 197–241 (Freeman and Co., New York, 1992).
Dobson, C.M., Evans, P.A. & Radford, S.E. Understanding how proteins fold: the lysozyme story so far. Trends. Biochem. Sci. 19, 31–37 (1994).
Woodruff, W.H., Dyer, R.B., Williams, S., Callender, R.H. & Gilmanshin, R. Fast events in protein folding and unfolding: time-resolved infrared study of structure changes in apomyoglobin and model peptides. Protein Sci. 4 (2) 68 (1995).
Santoro, M.M. & Bolen, D.W. Unfolding free energies determined by the linear extrapolation method, 1. Unfolding of phenylmethane sulfonyl α-chymotrypsin using different denaturants. Biochemistry 27, 8063–8068 (1988).
Tanford, C. Protein denaturation. Part C. Theoretical models for the mechanism of denaturation. Adv. Prot. Chem. 24, 2–95 (1970).
Chen, B., Baase, W.A., Schellman, J.A. Low-temperature unfolding of a mutant of phage T4 lysozyme. 2. Kinetic investigations. Biochemistry 27, 691–699 (1989).
Schellman, J.A. Selective binding and solvent denaturation. Biopolymers 26, 549–559 (1987).
Nishii, I., Kataoka, M. & Goto, Y. Thermodynamic stability of the molten globule states of apomyoglobin. J. Mol. Biol. 250, 223–238 (1995).
Pörschke, D. & Eigen, M. Cooperative Non-enzymatic base recognition. III. Kinetics of the helix-coil transition of the oligoribouridylic-oligoriboadenylic acid alone at acidic pH. J. Mol. Biol. 62, 361–381 (1971).
Pörschke, D. A direct measurement of the unzippering rate of a nucleic acid double helix. Biophys. Chem. 2, 97–101 (1974).
Tsong, T.Y., Baldwin, R.L. & McPhie, P. A sequential model of mucleation-dependent protein folding: kinetic studies of ribonuclease A. J. Mol. Biol. 63, 453–469 (1972).
Elson, E.L. Analysis of the steady-state approximation for the sequential model. J. Mol. Biol. 63, 469–475 (1972).
Fersht, A.R. Optimization of rates of protein folding: the nucleation-condensation mechanism and its implications. Proc. Natl. Acad. Sci. USA 92, 10869–10873 (1995).
Shakhnovich, E., Abkevich, V. & Ptitsyn, O. Conserved residues and the mechanism of proten folding. Nature 379, 96–98 (1996).
Gittis, A.G., Stites, W.E. & Lattman, E.E. The phase transition between a compact denatured state and a random coil state in staphylococcal nuclease is first-order. J. Mol. Biol. 232, 718–724 (1993).
Griko, Y.V., Griko Freire, E. & Privalov, P.L. Energetics of the α-lactalbumin states: a calorimetric and statistical thermodynamic study. Biochemistry 33, 1889–1899 (1994).
Griko, Y.V. & Privalov, P.L. Thermodynamic puzzle of apomyoglobin unfolding. J. Mol. Biol. 235, 1318–1325 (1994).
Ptitsyn, O.B. & Uversky, V.N. The molten globule is a third thermodynamical state of protein molecules. FEBS Lett. 341, 15–18 (1994).
Kiefhaber, T. & Baldwin, R.L. Intrinsic stability of individual α-helices modulates structure and stability of the apomyoglobin molten globule form. J. Mol. Biol. 252, 122–132 (1995).
Edelhoch, H. Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry 6, 1948–1954 (1967).
Tonomura, B., Nakatani, H., Ohnishi, M., Yamaguchi-lto, J. & Hiromi, K. Test reaction for a stopped-flow apparatus. Anal. Biochem. 84, 370–383 (1978).
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Jamin, M., Baldwin, R. Refolding and unfolding kinetics of the equilibrium folding intermediate of apomyoglobin. Nat Struct Mol Biol 3, 613–618 (1996). https://doi.org/10.1038/nsb0796-613
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DOI: https://doi.org/10.1038/nsb0796-613
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