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Parallel protein-unfolding pathways revealed and mapped

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Abstract

Theoretical studies of protein folding suggest that multiple folding pathways should exist, but there is little experimental evidence to support this. Here we demonstrate changes in the flux between different transition states on parallel folding pathways, resulting in unprecedented upward curvature in the denaturant-dependent unfolding kinetics of a β-sandwich protein. As denaturant concentration increases, the highly compact transition state of one pathway becomes destabilized and the dominant flux of protein molecules shifts toward another pathway with a less structured transition state. Furthermore, point mutations alter the relative accessibility of the pathways, allowing the structure of two transition states on separate, direct folding pathways to be mapped by systematic Φ-value analysis. It has been suggested that pathways with diffuse rather than localized transition states are evolutionarily selected to prevent misfolding, and indeed we find that the transition state favored at high concentrations of denaturant is more polarized than the physiologically relevant one.

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Figure 1: Unfolding rate constants versus denaturant concentration.
Figure 2: Unfolding rates for the two pathways.
Figure 3: Energy landscape for wild-type TI I27.
Figure 4: Transition states of pathways L and H.

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Change history

  • 13 July 2003

    Replaced math image, appended PDF with correction info

Notes

  1. *Note: In the version of this article initially published online, the parallel-pathway model appearing on page 2 contains a mistake. This mistake has been corrected for the HTML and print versions of the article.

References

  1. Onuchic, J.N., Luthey-Schulten, Z. & Wolynes, P.G. Theory of protein folding: the energy landscape perspective. Annu. Rev. Phys. Chem. 48, 545–600 (1997).

    Article  CAS  Google Scholar 

  2. Dobson, C.M., Sali, A. & Karplus, M. Protein folding: a perspective from theory and experiment. Angew. Chem. Int. Ed. 37, 868–893 (1998).

    Article  Google Scholar 

  3. Baldwin, R.L. The nature of protein folding pathways: the classical versus the new view. J. Biomol. NMR 5, 103–109 (1995).

    Article  CAS  Google Scholar 

  4. Wildegger, G. & Kiefhaber, T. Three-state model for lysozyme folding: triangular folding mechanism with an energetically trapped intermediate. J. Mol. Biol. 270, 294–304 (1997).

    Article  CAS  Google Scholar 

  5. Dinner, A.R., Sali, A., Smith, L.J., Dobson, C.M. & Karplus, M. Understanding protein folding via free-energy surfaces from theory and experiment. Trends Biochem. Sci. 25, 331–339 (2000).

    Article  CAS  Google Scholar 

  6. Zaidi, F.N., Nath, U. & Udgaonkar, J.B. Multiple intermediates and transition states during protein unfolding. Nat. Struct. Biol. 4, 1016–1023 (1997).

    Article  CAS  Google Scholar 

  7. Baldwin, R.L. Competing unfolding pathways. Nat. Struct. Biol. 4, 965–966 (1997).

    Article  CAS  Google Scholar 

  8. Fowler, S.B. & Clarke, J. Mapping the folding pathway of an immunoglobulin domain: structural detail from phi value analysis and movement of the transition state. Structure 9, 355–366 (2001).

    Article  CAS  Google Scholar 

  9. Fersht, A.R. Optimisation of rates of protein folding: the nucleation-condensation mechanism and its implications. Biochemistry 92, 10869–10873 (1995).

    CAS  Google Scholar 

  10. Tanford, C. Protein denaturation. B. The transition from native to denatured state. Adv. Protein Chem. 23, 218–282 (1968).

    Google Scholar 

  11. Oliveberg, M., Tan, Y.-J., Silow, M. & Fersht, A.R. The changing nature of the protein folding transition state: implications for the shape of the free-energy profile for folding. J. Mol. Biol. 277, 933–943 (1998).

    Article  CAS  Google Scholar 

  12. Hammond, G.S. A correlation of reaction rates. J. Am. Chem. Soc. 77, 334–338 (1955).

    Article  CAS  Google Scholar 

  13. Matoushchek, A. & Fersht, A.R. Application of physical organic chemistry to engineered mutants of proteins: Hammond postulate behavior in the transition state of protein folding. Proc. Natl. Acad. Sci. USA 90, 7814–7818 (1993).

    Article  Google Scholar 

  14. Sanchez, I.E. & Kiefhaber, T. Evidence for sequential barriers and obligatory intermediates in apparent two-state protein folding. J. Mol. Biol. 325, 367–376 (2002).

    Article  Google Scholar 

  15. Otzen, D.E., Kristensen, O., Proctor, M. & Oliveberg, M. Structural changes in the transition state of protein folding: alternative interpretations of curved chevron plots. Biochemistry 38, 6499–6511 (1999).

    Article  CAS  Google Scholar 

  16. Matthews, J.M. & Fersht, A.R. Exploring the energy surface of protein folding by structure-reactivity relationships and engineering proteins: observation of Hammond behavior for the gross structure of the transition state and anti-Hammond behavior for structural elements for unfolding/folding of barnase. Biochemistry 34, 6805–6814 (1995).

    Article  CAS  Google Scholar 

  17. Dalby, P.A., Oliveberg, M. & Fersht, A.R. Movement of the intermediate and rate determining state of barnase on the energy landscape with changing temperature. Biochemistry 37, 4674–4679 (1998).

    Article  CAS  Google Scholar 

  18. Daggett, V., Li, A. & Fersht, A.R. Combined molecular dynamics and Φ-value analysis of structure-reactivity relationships in the transition state and unfolding pathway of barnase: structural basis of Hammond and anti-Hammond effects. J. Am. Chem. Soc. 120, 12740–12754 (1998).

    Article  CAS  Google Scholar 

  19. Otzen, D.E. & Oliveberg, M. Conformational plasticity in folding of the split β–α–β protein S6: evidence for burst-phase disruption of the native state. J. Mol. Biol. 317, 613–627 (2002).

    Article  CAS  Google Scholar 

  20. Fersht, A.R., Itzhaki, L.S., ElMasry, N.F., Matthews, J.M. & Otzen, D.E. Single versus parallel pathways of protein folding and fractional formation of structure in the transition state. Proc. Natl. Acad. Sci. USA 91, 10426–10429 (1994).

    Article  CAS  Google Scholar 

  21. Connors, K.A. Chemical Kinetics (VCH, New York, 1990).

    Google Scholar 

  22. Tanford, C. Protein denaturation. C. Theoretical models for the mechanism of denaturation. Adv. Prot. Chem. 24, 1–95 (1970).

    CAS  Google Scholar 

  23. Myers, J. & Oas, T.G. Mechanisms of fast protein folding. Annu. Rev. Biochem. 71, 783–815 (2002).

    Article  CAS  Google Scholar 

  24. Sanchez, I.E. & Kiefhaber, T. Hammond behavior versus ground state effects in protein folding: evidence for narrow free energy barriers and residual structure in unfolded states. J. Mol. Biol. 327, 867–884 (2003).

    Article  CAS  Google Scholar 

  25. Fersht, A.R. Structure and Mechanism in Protein Science (W.H. Freeman, New York, 1998).

    Google Scholar 

  26. McCallister, E.L., Alm, E. & Baker, D. Critical role of β-hairpin formation in protein G. Nat. Struct. Biol. 7, 669–673 (2000).

    Article  CAS  Google Scholar 

  27. Nauli, S., Kuhlman, B. & Baker, D. Computer-based redesign of a protein folding pathway. Nat. Struct. Biol. 8, 602–605 (2001).

    Article  CAS  Google Scholar 

  28. Fersht, A.R., Matouschek, A. & Serrano, L. The folding of an enzyme I. Theory of protein engineering analysis of stability and pathway of protein folding. J. Mol. Biol. 224, 771–782 (1992).

    Article  CAS  Google Scholar 

  29. Lindberg, M., Tangrot, J. & Oliveberg, M. Complete change of the protein folding transition state upon circular permutation. Nat. Struct. Biol. 9, 818–822 (2002).

    CAS  PubMed  Google Scholar 

  30. Davis, R., Dobson, C.M. & Vendruscolo, M. Determination of the structures of distinct transition state ensembles for a β-sheet peptide with parallel folding pathways. J. Chem. Phys. 117, 9510–9517 (2002).

    Article  CAS  Google Scholar 

  31. Carrion-Vasquez, M. et al. Mechanical and chemical unfolding of a single protein: a comparison. Proc. Natl. Acad. Sci. USA 96, 3694–3699 (1999).

    Article  Google Scholar 

  32. 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 

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Acknowledgements

We thank C. Dobson for support and encouragement and S. Fowler and A. Fersht for helpful discussions. This work was supported by the Wellcome Trust (C.F.W., L.R. and J.C.). J.C. is a Wellcome Trust senior research fellow and C.F.W. holds a Wellcome Trust prize studentship.

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Correspondence to Jane Clarke.

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Wright, C., Lindorff-Larsen, K., Randles, L. et al. Parallel protein-unfolding pathways revealed and mapped. Nat Struct Mol Biol 10, 658–662 (2003). https://doi.org/10.1038/nsb947

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