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Kinetic intermediates in the formation of the cytochrome c molten globule

Nature Structural Biology volume 3, pages 1019–1025 (1996)Cite this article

Abstract

The relationship between molten globules and transient intermediates in protein folding has been explored by equilibrium and kinetic analysis of the compact acid-denatured A-state of cytochrome c. The chloride-induced formation of the A-state is a complex reaction with structural intermediates resembling those found under native refolding conditions, including a rapidly formed compact state and a subsequent intermediate with interacting N- and C-terminal helices. Together with mutational evidence for specific helix–helix packing interactions, this shows that the A-state is a stable analogue of a late folding intermediate. The L94A mutation blocks all folding steps after the initial collapse and its equilibrium state resembles early kinetic intermediates.

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References

  1. Dobson, C.M. Solid evidence for molten globules. Curr. Biol. 4, 636–640 (1994).

    Article  CAS  Google Scholar 

  2. Ptitsyn, O.B. Molten globule and protein folding. Adv. Prot. Chem. 47, 83–229 (1995).

    CAS  Google Scholar 

  3. Baum, J., Dobson, C.M., Evans, P.A. & Hanley, C. Characterization of a partly folded protein by NMR methods: Studies on the molten globule state of Guinea pig α-lactalbumin. Biochemistry 28, 7–13 (1989).

    Article  CAS  Google Scholar 

  4. Wu, L.C., Peng, Z.-y. & Kim, P.S. Bipartite structure of the α-lacatalbumin molten globule. Nature Struct. Biology 2, 281–286 (1995).

    Article  CAS  Google Scholar 

  5. Kuwajima, K. The molten globule state of α-lactalbumin. FASEB J. 10, 102–109 (1996).

    Article  CAS  Google Scholar 

  6. Hughson, F.M., Wright, P.E. & Baldwin, R.L. Structural characterization of a partly folded apomyoglobin intermediate. Science 249, 1544–1548 (1990).

    Article  CAS  Google Scholar 

  7. Kay, M.S. & Baldwin, R.L. Packing interactions in the apomyoglobin folding intermediate. Nature Struct. Biology 3, 439–445 (1996).

    Article  CAS  Google Scholar 

  8. 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  Google Scholar 

  9. 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  Google Scholar 

  10. Baldwin, R.L. Molten globules: Specific or nonspecific folding intermediates. Biochem. Mol. Biol. 2, 379–389 (1991).

    CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  12. Roder, H. & Elöve, G.A. Early stages of protein folding. In Mechanisms of Protein Folding: Frontiers in Molecular Biology. (ed. R.H. Pain) 26–55 (Oxford University Press, New York; 1994).

    Google Scholar 

  13. Evans, P.A. & Radford, S.E. Probing the structure of folding intermediates. Curr. Opin. Struct. Biol. 4, 100–106 (1994).

    Article  CAS  Google Scholar 

  14. Fink, A.L. Compact intermediate states in protein folding. Annu. Rev. Biophys. Biomol. Struct. 24, 495–522 (1995).

    Article  CAS  Google Scholar 

  15. Jennings, P.A. & Wright, P.E. Formation of a molten globule intermediate early in the kinetic folding pathway of apomyoglobin. Science 262, 892–895 (1993).

    Article  CAS  Google Scholar 

  16. Balbach, J. et al. Following protein folding in real time using NMR spectroscopy. Nature Struct. Biology 2, 865–870 (1995).

    Article  CAS  Google Scholar 

  17. 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  Google Scholar 

  18. Robinson, J.B.J., Strottmann, J.M. & Stellwagen, E. A globular high spin form of ferricytochrome c. J. Biol. Chem. 258, 6772–6776 (1983).

    CAS  PubMed  Google Scholar 

  19. Potekhin, S. & Pfeil, W. Microcalorimetric studies of conformational transitions of f erricytochrome c in acidic solution. Biophysical Chemistry 34, 55–62 (1989).

    Article  CAS  Google Scholar 

  20. 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  Google Scholar 

  21. Goto, Y., Calciano, L.J. & Fink, A.L. Acid-induced folding of proteins. Proc. Natl. Acad. Sci. USA 87, 573–577 (1990).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  23. Kuroda, Y., Kidokoro, S. & Wada, A. Thermodynamic characterization of cytochrome c at low pH. J. Mol. Biol. 223, 1139–1153 (1992).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  25. Hagihara, Y., Tan, Y. & Goto, Y. Comparison of the conformational stability of the molten globule and native states of horse cytochrome c. J. Mol. Biol. 237, 336–348 (1994).

    Article  CAS  Google Scholar 

  26. Chalikian, T.V., Gindikin, V.S. & Breslauer, K.J. Volumetric characterizations of the native, molten globule and unfolded states of cytochrome c at acidic pH. J. Mol. Biol. 250, 291–306 (1995).

    Article  CAS  Google Scholar 

  27. Kuroda, Y., Endo, S., Nagayama, K. & Wada, A. Stability of α-helices in a molten globule state of cytochrome c by hydrogen-deuterium exchange and two-dimensional NMR spectroscopy. J. Mol. Biol. 247, 682–688 (1995).

    CAS  PubMed  Google Scholar 

  28. Jordan, T., Eads, J.C. & Spiro, T.G. Secondary and tertiary structure of the A-state of cytochrome c from resonance Raman spectroscopy. Protein Sci. 4, 716–728 (1995).

    Article  CAS  Google Scholar 

  29. Marmorino, J.L. & Pielak, G.J. A native tertiary interaction stabilizes the A state of cytochrome c. Biochemistry 34, 3140–3143 (1995).

    Article  CAS  Google Scholar 

  30. Dyson, H.J. & Beattie, J.K. Spin state and unfolding equilibria of ferricytochromec in acidic solutions. J. Biol. Chem. 257, 2267–2273 (1982).

    CAS  PubMed  Google Scholar 

  31. Hamada, D. et al. Role of heme axial ligands in the conformational stability of the native and molten globule states of horse cytochrome c. J. Mol. Biol. 256, 172–186 (1996).

    Article  CAS  Google Scholar 

  32. Goto, Y. & Nishikiori, S. Role of electrostatic repulsion in the acidic molten globule of cytochrome c. J. Mol. Biol. 222, 679–686 (1991).

    Article  CAS  Google Scholar 

  33. Jamin, M. & Baldwin, R.L. Refolding and unfolding kinetics of the equilibrium molten globule intermediate of apomyoglobin. Nature Struct. Biology 3, 613–618 (1996).

    Article  CAS  Google Scholar 

  34. Sosnick, T.R., Mayne, L., Hiller, R. & Englander, S.W. The barriers in protein folding. Nature Struct. Biology 1, 149–156 (1994).

    Article  CAS  Google Scholar 

  35. Colón, W., Elöve, G.A., Wakem, L.P., Sherman, F. & Roder, H. Side chain packing of the N- and C-terminal helices plays a critical role in the kinetics of cytochrome c folding. Biochemistry 35, 5538–5549 (1996).

    Article  Google Scholar 

  36. Stellwagen, E. & Babul, J. Stabilization of the globular structure of ferricytochrome c by chloride in acidic solvents. Biochemistry 14, 5135–5140 (1975).

    Article  CAS  Google Scholar 

  37. 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  Google Scholar 

  38. Misra, V.K., Sharp, K.A., Friedman, R.A. & Honig, B. Salt effects on ligand-DNA binding. J. Mol. Biol. 238, 245–263 (1994).

    Article  CAS  Google Scholar 

  39. Fersht, A. Enzyme structure and mechanism. (W.H. Freeman and company, New York; 1985).

    Google Scholar 

  40. 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  Google Scholar 

  41. 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  Google Scholar 

  42. Elöve, G.A. & Roder, H. Structure and stability of cytochrome c folding intermediates. ACS Symposium Series 470, 50–63 (1991).

    Article  Google Scholar 

  43. Elöve, G.A., Bhuyan, A.K. & Roder, H. Kinetic mechanism of cytochrome c folding: involvement of the heme and its ligands. Biochemistry 33, 6925–6935 (1994).

    Article  Google Scholar 

  44. Bai, Y., Sosnick, T.R., Mayne, L. & Englander, S.W. Protein folding intermediates: native-state hydrogen exchange. Science 269, 192–197 (1995).

    Article  CAS  Google Scholar 

  45. Babul, J. & Stellwagen, E. Participation of the protein ligands in the folding of cytochrome c. Biochemistry 11, 1195–1200 (1972).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  47. Uversky, V.N. & Ptitsyn, O.B. “Partly folded” state, a new equilibrium state of protein molecules: Four-state guanidinium chloride-induced unfolding of β-lactamase at low temperature. Biochemistry 33, 2782–2791 (1994).

    Article  CAS  Google Scholar 

  48. Uversky, V.N. & Ptitsyn, O.B. Further evidence on the equilibrium “pre-molten globule state”: four-state guanidinium chloride-induced unfolding of carbonic anhydrase B at low temperature. J. Mol. Biol. 255, 215–228 (1996).

    Article  CAS  Google Scholar 

  49. Khorasanizadeh, S., Peters, I.D. & Roder, H. Evidence for a three-state model of protein folding from kinetic analysis of ubiquitin variants with altered core residues. Nature Struct. Biology 3, 193–205 (1996).

    Article  CAS  Google Scholar 

  50. Sosnick, T.R., Mayne, L. & Englander, S.W. Molecular collapse: the rate-limiting step in two-state cytochrome c folding. Proteins 24, 413–426 (1996).

    Article  CAS  Google Scholar 

  51. Tsong, T.Y. The Trp-59 fluorescence of ferricytochrome c as a sensitive measure of the over-all protein conformation. J. Biol. Chem. 249, 1988–1990 (1974).

    CAS  PubMed  Google Scholar 

  52. Scholtz, J.M. & Baldwin, R.L. The mechanism of alpha-helix formation by peptides. Annu. Rev. Biophys. Biomol. Struct. 21, 95–118 (1992).

    Article  CAS  Google Scholar 

  53. Chan, H.S. & Dill, K.A. Origins of structure in globular proteins. Proc. Natl. Acad. Sci. USA 87, 6388–6392 (1990).

    Article  CAS  Google Scholar 

  54. Agashe, V.R., Shastry, M.C.R. & Udgaonkar, J.B. Initial hydrophobic collapse in the folding of barstar. Nature 377, 754–757 (1995).

    Article  CAS  Google Scholar 

  55. Privalov, P.L. Intermediate states in protein folding. J. Mol. Biol. 258, 707–725 (1996).

    Article  CAS  Google Scholar 

  56. Khorasanizadeh, S., Peters, I.D., Butt, T.R. & Roder, H. Stability and folding of a tryptophan-containing mutant of ubiquitin. Biochemistry 32, 7054–7063 (1993).

    Article  CAS  Google Scholar 

  57. Bushnell, G.W., Louie, G.V. & Brayer, G.D. High-resolution three-dimensional structure of horse heart cytochrome c. J. Mol. Biol. 214, 585–595 (1990).

    Article  CAS  Google Scholar 

  58. Kraulis, P.J. MOLSCRIPT: a program to produce both detailed and schematic plots of protein structures. J. Appl. Cryst. 24, 946–950 (1991).

    Article  Google Scholar 

  59. Pace, C.N. Determination and analysis of urea and guanidine hydrochloride denaturation curves. Meth. Enzymol. 131, 266–280 (1986).

    Article  CAS  Google Scholar 

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Authors and Affiliations

  1. Institute for Cancer Research, Fox Chase Cancer Center, 7701 Burholme Ave., Philadelphia, Pennsylvania, 19111, USA

    Wilfredo Colón & Heinrich Roder

  2. Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania, 19104, USA

    Heinrich Roder

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  1. Wilfredo Colón
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  2. Heinrich Roder
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Colón, W., Roder, H. Kinetic intermediates in the formation of the cytochrome c molten globule. Nat Struct Mol Biol 3, 1019–1025 (1996). https://doi.org/10.1038/nsb1296-1019

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