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The barriers in protein folding

Nature Structural Biology volume 1, pages 149–156 (1994)Cite this article

Abstract

Elimination of an interaction which forms in denatured cytochrome c enables the majority of the molecules to fold to the native state on a 15 ms time scale, without populating observable intermediates. These results are contrary to the current view that particular steps in protein folding, including the supposedly rate–limiting molten globule to native transition, are intrinsically slow. Instead it appears that intermediates characterized so far may be kinetically trapped by barriers that are optional rather than integral to the folding process. Major barriers may result from misorganization of the chain in the initial condensation step.

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References

  1. Kuwajima, K. The molten globule state as a clue for understanding the folding and cooperativity of globular-protein structure. Proteins Struct. Funct. Genet. 6, 87–103 (1989).

    Article  CAS  PubMed  Google Scholar 

  2. Kim, P.S. & Baldwin, R.L. Intermediates in the folding reactions of small proteins. Annu. Rev. Biochem. 59, 631–660 (1990).

    Article  CAS  PubMed  Google Scholar 

  3. Ptitsyn, O.B. & Semisotnov, G.V. The mechanism of protein folding. in Conformations and Forces in Protein Folding (eds Nail, B. T. & Dill, K.A.) 155–168 (AAAS, Washington DC, (1991).

    Google Scholar 

  4. Chan, H.S. & Dill, K.A. The protein folding problem. Physics Today 46, 24–32 (1993).

    Article  CAS  Google Scholar 

  5. Baldwin, R.L., Pulsed H/D-exchange studies of folding intermediates. Curr. Opin. struct. Biol. 3, 84–91 (1993).

    Article  CAS  Google Scholar 

  6. Matthews, C.R. Pathways of protein folding. A. Rev. Biochem. 62, 653–683 (1993).

    Article  CAS  Google Scholar 

  7. Brems, D.N. & Stellwagen, E. Manipulation of the observed kinetic phases in the refolding of denatured ferricytochromes c. J. biol. Chem. 258, 3655–3660 (1983).

    CAS  PubMed  Google Scholar 

  8. Udgaonkar, J.B. & Baldwin, R.L. Early folding intermediate of ribonuclease A. Proc. natn. Acad. Sci. U.S.A. 87, 8197–8201 (1990).

    Article  CAS  Google Scholar 

  9. Jackson, S.E. & Fersht, A.R. Folding of chymotrypsin inhibitor 2. 1. Evidence for a two-state transition. Biochemistry 30, 10428–10435 (1991).

    Article  CAS  PubMed  Google Scholar 

  10. Alexander, P., Orban, J. & Bryan, P. Kinetic analysis of folding and unfolding of the 56 amino acid IgG-binding domain of streptococcal protein G. Biochemistry 31, 7243–7248 (1992).

    Article  CAS  PubMed  Google Scholar 

  11. Briggs, M.S. & Roder, H. Early hydrogen-bonding events in the folding reaction of ubiquitin. Proc. natn. Acad. Sci. U.S.A. 89, 2017–2021 (1992).

    Article  CAS  Google Scholar 

  12. Radford, S.E., Dobson, C.M. & Evans, P.A. The folding of hen lysozyme involves partially structured intermediates and multiple pathways. Nature 358, 302–307 (1992).

    Article  CAS  PubMed  Google Scholar 

  13. Miranker, A., Robinson, C.V., Radford, S.E., Aplin, R.T. & Dobson, C.M. Detection of transient protein folding populations by mass spectrometry. Science 262, 896–900 (1993).

    Article  CAS  PubMed  Google Scholar 

  14. Roder, H., Elöve, G.A. & Englander, S.W. Structural characterization of folding intermediates in cytochrome c by H- exchange labelling and proton NMR. Nature 335, 700–704 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Elöve, G.A. & Roder, H. Structure and stability of cytochrome c folding intermediates. In Protein Refolding (eds Georgiou, G. & De Bernardez-Clark, E.) 50–63 (ACS Symposium Series, Washington DC, 1991).

    Chapter  Google Scholar 

  16. Elöve, G.A., Chaffotte, A.F., Roder, H. & Goldberg, M.E. Early steps in cytochrome c folding probed by time-resolved circular dichroism and fluorescence spectroscopy. Biochemistry 31, 6876–6883 (1992).

    Article  PubMed  Google Scholar 

  17. Tsong, T.Y. Ferricytochrome c chain folding measured by the energy transfer of tryptophan 59 to the heme group. Biochemistry 15, 5467–5473 (1976).

    Article  CAS  PubMed  Google Scholar 

  18. Myer, Y.P., Pande, A. & Saturno, A.F. Kinetics of unfolding andfolding of horse heart ferricytochrome c with urea. J. biol. Chem. 256, 1576–1581 (1981).

    CAS  PubMed  Google Scholar 

  19. Ridge, J.A., Baldwin, R.L. & Labhardt, A.M. Nature of the fast and slow refolding reactions of iron(III) cytochrome c. Biochemistry 20, 1622–1630 (1981).

    Article  CAS  PubMed  Google Scholar 

  20. Nall, B.T. Structural intermediates in folding of yeast iso-2 cytochrome c. Biochemistry 22, 1423–1429 (1983).

    Article  CAS  PubMed  Google Scholar 

  21. Muthukrishnan, K. & Nall, B.T. Effective concentrations of amino acid side chains in an unfolded protein. Biochemistry 30, 4706–4710 (1991).

    Article  CAS  PubMed  Google Scholar 

  22. Tsong, T.Y. An acid induced conformational transition of denatured cytochrome c in urea and guanidine hydrochloride solutions. Biochemistry 14, 1542–1547 (1975).

    Article  CAS  PubMed  Google Scholar 

  23. Dyson, H.J. & Beattie, J.K. Spin state and unfolding equilibria of ferricytochrome c in acidic solutions. J. biol. Chem. 257, 2267–2273 (1982).

    CAS  PubMed  Google Scholar 

  24. Schechter, E. & Saludjian, P. Conformation of ferricytochrome c. IV. Relationship between optical absorption and protein conformation. Biopolymers 5, 788–790 (1967).

    Article  Google Scholar 

  25. Myer, Y.P. Ferricytochrome c. Refolding and the methionine 80-sulfur-iron linkage. J. biol. Chem. 259, 6127–6133 (1984).

    CAS  PubMed  Google Scholar 

  26. Udgaonkar, J.B. & Baldwin, R.L. NMR evidence for an early framework intermediate on the folding pathway of ribonuclease A. Nature 335, 694–699 (1988).

    Article  CAS  PubMed  Google Scholar 

  27. Englander, S.W. & Mayne, L. Protein folding studied using hydrogen-exchange labeling and two-dimensional NMR. A. Rev. Biophys. biomol. Struct. 21, 243–265 (1992).

    Article  CAS  Google Scholar 

  28. Varley, P. et al. Kinetics of folding of the all-ß sheet protein interleukin-1β. Science 260, 1110–1113 (1993).

    Article  CAS  PubMed  Google Scholar 

  29. Nall, B.T. Native or nativelike species are transient intermediates in folding of alkaline iso-2 cytochrome c. Biochemistry 25, 2974–2978 (1986).

    Article  CAS  PubMed  Google Scholar 

  30. Bushnell, G.W., Louie, G.V. & Brayer, G.D. High-resolution three dimensional structure of horse heart cytochrome c. J. molec. Biol. 213, 585–595 (1990).

    Article  Google Scholar 

  31. Schmid, F.X., Buonocore, M.H. & Baldwin, R.L. Tests of the simple model of Lin and Brandts for the folding kinetics of ribonuclease A. Biochemistry 3389–3394 (1984).

    Article  CAS  PubMed  Google Scholar 

  32. Schultz, D.A., Schmid, F.X. & Baldwin, R.L. Cis proline mutants of ribonuclease A. II. Elimination of the slow-folding forms by mutation. Protein Science 1, 917–924 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Kiefhaber, T. & Schmid, F.X. Kinetic coupling between protein folding and prolyl isomerization. II. Folding of ribonuclease A and ribonuclease T1. J. molec. Biol. 224, 231–240 (1992).

    Article  CAS  PubMed  Google Scholar 

  34. Mullins, L.S., Pace, C.N. & Raushel, F.M. Investigation of ribonuclease T1 folding intermediates by hydrogen-deuterium amide exchange-two-dimensional NMR spectroscopy. Biochemistry 32, 6152–6156 (1993).

    Article  CAS  PubMed  Google Scholar 

  35. Ptitsyn, O.B., Pain, R.H., Semisotnov, G.V., Zerovnik, E. & Razgulyaev, O.I. Evidence for a molten globule state as a general intermediate in protein folding. FEBS Lett. 262, 20–24 (1990).

    Article  CAS  PubMed  Google Scholar 

  36. 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).

    Article  CAS  PubMed  Google Scholar 

  37. Ohgushi, M. & Wada, A. Molten-globule state: a compact form of globular proteins with mobile side-chains. FEBS Lett. 164, 21–24 (1983).

    Article  CAS  PubMed  Google Scholar 

  38. Ptitsyn, O.B. Protein folding: hypotheses and experiments. J. Protein Chem. 6, 273–293 (1987).

    Article  CAS  Google Scholar 

  39. Dill, K.A. & Shortle, D. Denatured states of proteins. A. Rev. Biochemistry 60, 795–825 (1991).

    Article  CAS  Google Scholar 

  40. Haynie, D.T. & Freire, E. Structural energetics of the molten globule state. Proteins Struct. Func. Genet. 16, 115–140 (1993).

    Article  CAS  Google Scholar 

  41. Fink, A.L., Molten Globules. in Methods in molecular biology: Protein stability and folding protocols (ed. Shirley, B. A.) (Humana Press, Clifton, in the press).

  42. Jeng, M.F. & Englander, S.W. Stable submolecular folding units in a non-compact form of cytochrome c. J. molec. Biol. 221, 1045–1061 (1991).

    Article  CAS  PubMed  Google Scholar 

  43. Kataoka, M., Hagihara, Y., Mihara, K. & Goto, Y. Molten globule of cytochrome c studied by small angle X-ray scattering. J. molec. Biol. 229, 591–596 (1993).

    Article  CAS  PubMed  Google Scholar 

  44. Kuroda, Y., Kidokoro, S. & Wada, A. Thermodynamic characterization of cytochrome c at low pH: Observation of the molten globule state and of the cold denaturation process. J. molec. Biol. 223, 1139–1153 (1992).

    Article  CAS  PubMed  Google Scholar 

  45. Jeng, M.F., Englander, S.W., Elöve, G.A., Wand, A.J. & Roder, H. Structural description of acid-denatured cytochrome c by hydrogen exchange and 2D NMR. Biochemistry 29, 10433–10437 (1990).

    Article  CAS  PubMed  Google Scholar 

  46. Kuwajima, K., Hiraoka, Y., Ikeguchi, M. & Sugai, S. Comparison of the transient folding intermediates of lysozyme and α-lactalbumin. Biochemistry 24, 874–881 (1985).

    Article  CAS  PubMed  Google Scholar 

  47. Alexandrescu, A.T., Evans, P.A., Pitkeathly, M., Baum, J. & Dobson, C.M. Structure and dynamics of the acid-denatured molten globulestate of α-lactalbumin: A two-dimensional NMR study. Biochemistry 32, 1707–1718 (1993).

    Article  CAS  PubMed  Google Scholar 

  48. Barrick, D. & Baldwin, R.L. The molten globule intermediate of apomyoglobin and the process of protein folding. Protein Science 2, 869–876 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Eliezer, D. et al. Evidence of an associative intermediate on the myoglobin refolding pathway. Biophys. J. 65, 912–917 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Dill, K.A., Fiebig, K.M. & Chan, H.S. Cooperativity in protein-folding kinetics. Proc. natn. Acad. Sci. U.S.A. 90, 1942–1946 (1993).

    Article  CAS  Google Scholar 

  51. Texter, F.L., Spencer, D.B., Rosenstein, R. & Matthews, C.R. Intramolecular catalysis of a proline isomerization reaction in the folding of dihyrofolate reductase. Biochemistry 31, 5687–5691 (1992).

    Article  CAS  PubMed  Google Scholar 

  52. Kiefhaber, T., Grunert, H.P., Hahn, U . & Schmid, F.X. Folding of RNase T1 is decelerated by a specific tertiary contact in a folding intermediate. Proteins Struct. Funct. Genet. 12, 171–179 (1992).

    Article  CAS  PubMed  Google Scholar 

  53. Schreiber, G. & Fersht, A.R. The refolding of cis- and trans-peptidylprolyl isomers of barstar. Biochemistry 32, 11195–11203 (1993).

    Article  CAS  PubMed  Google Scholar 

  54. Mann, C.J. & Matthews, C.R. Structure and stability of an early folding intermediate of E. coli trp aporepressor measured by far-UV stopped-flow circular dichroism and ANS binding. Biochemistry 32, 5282–5290(1993).

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Department of Biochemistry and Biophysics, Johnson Research Foundation, University of Pennsylvania, Philadelphia, Pennsylvania, 19104-6059

    T.R. Sosnick, L. Mayne, R. Hiller & S.W. Englander

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Sosnick, T., Mayne, L., Hiller, R. et al. The barriers in protein folding. Nat Struct Mol Biol 1, 149–156 (1994). https://doi.org/10.1038/nsb0394-149

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