Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Im7 folding mechanism: misfolding on a path to the native state

Nature Structural Biology volume 9, pages 209–216 (2002)Cite this article

Abstract

Many proteins populate collapsed intermediate states during folding. In order to elucidate the nature and importance of these species, we have mapped the structure of the on-pathway intermediate of the four-helix protein, Im7, together with the conformational changes it undergoes as it folds to the native state. Kinetic data for 29 Im7 point mutants show that the intermediate contains three of the four helices found in the native structure, packed around a specific hydrophobic core. However, the intermediate contains many non-native interactions; as a result, hydrophobic interactions become disrupted in the rate-limiting transition state before the final helix docks onto the developing structure. The results of this study support a hierarchical mechanism of protein folding and explain why the misfolding of Im7 occurs. The data also demonstrate that non-native interactions can play a significant role in folding, even for small proteins with simple topologies.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Ribbon diagram of Im7.
Figure 2: Folding and unfolding data for wild type Im7*.
Figure 3: Folding and unfolding data for A77G and V36A.
Figure 4: Folding and unfolding data for V16A and I72V.
Figure 5: Folding and unfolding data for T51S and I54A.
Figure 6: Folding and unfolding data for L34A and F41L.
Figure 7: Non-native interactions in the Im7 intermediate.

Similar content being viewed by others

References

  1. Fersht, A.R., Matouschek, A. & Serrano, L. The folding of an enzyme.1. Theory of protein engineering analysis of stability and pathway of protein folding. J. Mol. Biol. 224, 771–782 (1992).

    Article  CAS  Google Scholar 

  2. Fersht, A.R. Mapping the structures of transition-states and intermediates in folding — delineation of pathways at high-resolution. Phil. Trans. R. Soc. Lond. B 348, 11–15 (1995).

    Article  CAS  Google Scholar 

  3. Jackson, S.E. How do small single-domain proteins? Folding Des. 3, R81–R91 (1998).

    Article  CAS  Google Scholar 

  4. Grantcharova, V., Alm, E., Baker, D. & Horwich, A. Mechanisms of protein folding. Curr. Opin. Struct. Biol. 11, 70–82 (2001).

    Article  CAS  Google Scholar 

  5. Plaxco, K.W., Simons, K.T. & Baker, D. Contact order, transition state placement and the refolding rates of single domain proteins. J. Mol. Biol. 277, 985–994 (1998).

    Article  CAS  Google Scholar 

  6. Baker, D. A surprising simplicity to protein folding. Nature 405, 39–42 (2000).

    Article  CAS  Google Scholar 

  7. Yeh, S. & Rousseau, D.L. Hierarchical folding of cytochrome c. Nature Struct. Biol. 8, 689–694 (2000).

    Google Scholar 

  8. Roder, H. & Colón, W. Kinetic role of early intermediates in protein folding. Curr. Opin. Struct. Biol. 7, 15–28 (1997).

    Article  CAS  Google Scholar 

  9. Gunasekaran, K., Eyles, S.J., Hagler, A.T. & Gierasch, L.M. Keeping it in the family: folding studies of related proteins. Current Opin. Struct. Biol. 11, 83–93 (2001).

    Article  CAS  Google Scholar 

  10. Park, S.-H., Shastry, M.C.R. & Roder, H. Folding dynamics of the B1 domain of protein G explored by ultrarapid mixing. Nature Struct. Biol. 6, 943–947 (1999).

    Article  CAS  Google Scholar 

  11. Shastry, M.C. & Roder, H. Evidence for barrier-limited protein folding kinetics on the microsecond time scale. Nature Struct. Biol. 5, 385–392 (1998).

    Article  CAS  Google Scholar 

  12. Raschke, T.M., Kho, J. & Marqusee, S. Confirmation of the hierarchical folding of RNase H: a protein engineering study. Nature Struct. Biol. 6, 825–831 (1999).

    Article  CAS  Google Scholar 

  13. Sosnick, T.R., Shtilerman, M.D., Mayne, L. & Englander, S.W. Ultrafast signals in protein folding and the polypeptide contracted state. Proc. Natl. Acad. Sci. USA 94, 8545–8550 (1997).

    Article  CAS  Google Scholar 

  14. Qi, P.X., Sosnick, T.R. & Englander, S.W. The burst phase in ribonuclease A folding and solvent dependence of the unfolded state. Nature Struct. Biol. 5, 882–884 (1998).

    Article  CAS  Google Scholar 

  15. Mok, Y.K., Kay, C.M., Kay, L.E. & Forman-Kay, J. NOE data demonstrating a compact unfolded state for an SH3 domain under non-denaturing conditions. J. Mol. Biol. 289, 619–638 (1999).

    Article  CAS  Google Scholar 

  16. Baldwin, R.L. Folding consensus? Nature Struct. Biol. 8, 92–94. (2001).

    Article  CAS  Google Scholar 

  17. James, R., Kleanthous, C. & Moore, G.R. The biology of E colicins: Paradigms and paradoxes. Microbiology 142, 1569–1580 (1996).

    Article  CAS  Google Scholar 

  18. Ferguson, N., Capaldi, A.P., James, R., Kleanthous, C. & Radford, S.E. Rapid folding with and without populated intermediates in the homologous four-helix proteins Im7 and Im9. J. Mol. Biol. 286, 1597–1608 (1999).

    Article  CAS  Google Scholar 

  19. Gorski, S.A., Capaldi, A.P., Kleanthous, C. & Radford, S.E. Acidic conditions stabilise intermediates populated during the folding of Im7 and Im9. J. Mol. Biol. 312, 849–863 (2001).

    Article  CAS  Google Scholar 

  20. Capaldi, A.P., Shastry, M.C., Kleanthous, C., Roder, H. & Radford, S.E. Ultrarapid mixing experiments reveal that Im7 folds via an on-pathway intermediate. Nature Struct. Biol. 8, 68–72. (2001).

    Article  CAS  Google Scholar 

  21. Ozkan, S.B., Bahar, I. & Dill, K.A. Transition states and the meaning of Φ-values in protein folding kinetics. Nature Struct. Biol. 8, 765–769. (2001).

    Article  CAS  Google Scholar 

  22. Fersht, A.R. Characterizing transition-states in protein-folding — an essential step in the puzzle. Curr. Opin. Struct. Biol. 5, 79–84 (1995).

    Article  CAS  Google Scholar 

  23. Muñoz, V. & Serrano, L. Elucidating the folding problem of helical peptides using empirical parameters. 2. Helix macrodipole effects and rational modification of the helical content of natural peptides. J. Mol. Biol. 245, 275–296 (1995).

    Article  Google Scholar 

  24. Muñoz, V. & Serrano, L. Elucidating the folding problem of helical peptides using empirical parameters. 3. Temperature and pH-dependence. J. Mol. Biol. 245, 297–308 (1995).

    Article  Google Scholar 

  25. Muñoz, V. & Serrano, L. Development of the multiple sequence approximation within the AGADIR model of α–helix formation: Comparison with Zimm-Bragg and Lifson-Roig formalisms. Biopolymers 41, 495–509 (1997).

    Article  Google Scholar 

  26. Lacroix, E., Viguera, A.R. & Serrano, L. Elucidating the folding problem of α-helices: local motifs, long-range electrostatics, ionic strength dependence and prediction of NMR parameters. J. Mol. Biol. 284, 173–191 (1998).

    Article  CAS  Google Scholar 

  27. Kuhlmann, U.C., Pommer, A.J., Moore, G.R., James, R. & Kleanthous, C. Specificity in protein–protein interactions: The structural basis for dual recognition in endonuclease colicin–immunity protein complexes. J. Mol. Biol. 301, 1163–1178 (2000).

    Article  CAS  Google Scholar 

  28. Wallis, R. et al. Specificity in protein–protein recognition: conserved Im9 residues are the major determinants of stability in the colicin E9 DNase–Im9 complex. Biochemistry 37, 476–485 (1998).

    Article  Google Scholar 

  29. Li, W. et al. Dual recognition and the role of specificity-determining residues in colicin E9 DNase–immunity protein interactions. Biochemistry 37, 11771–11779 (1998).

    Article  CAS  Google Scholar 

  30. Baldwin, R.L. & Rose, G.D. Is protein folding hierarchic? I. Local structure and peptide folding. Trends Biochem. Sci. 24, 26–33 (1999).

    Article  CAS  Google Scholar 

  31. Baldwin, R.L. & Rose, G.D. Is protein folding hierarchic? II. Folding intermediates and transition states. Trends Biochem. Sci. 24, 77–83 (1999).

    Article  CAS  Google Scholar 

  32. Karplus, M. & Weaver, D.L. Protein folding dynamics — the diffusion-collision model and experimental-data. Protein Sci. 3, 650–668 (1994).

    Article  CAS  Google Scholar 

  33. Karplus, M. & Weaver, D.L. Protein-folding dynamics. Nature 260, 404–406 (1976).

    Article  CAS  Google Scholar 

  34. Burton, R.E., Myers, J.K. & Oas, T.G. Protein folding dynamics: quantitative comparison between theory and experiment. Biochemistry 37, 5337–5343 (1998).

    Article  CAS  Google Scholar 

  35. Myers, J.K. & Oas, T.G. Reinterpretation of GCN4-p1 folding kinetics: partial helix formation precedes dimerization in coiled coil folding. J. Mol. Biol. 289, 205–209 (1999).

    Article  CAS  Google Scholar 

  36. Myers, J.K. & Oas, T.G. Preorganized secondary structure as an important determinant of fast protein folding. Nature Struct. Biol. 8, 552–558. (2001).

    Article  CAS  Google Scholar 

  37. Pappu, R.V. & Weaver, D.L. The early folding kinetics of apomyoglobin. Protein Sci. 7, 480–490 (1998).

    Article  CAS  Google Scholar 

  38. Ko, T.P., Liao, C.C., Ku, W.Y., Chak, K.F. & Yuan, H.S. The crystal structure of the DNase domain of colicin E7 in complex with its inhibitor Im7 protein. Structure Fold. Des . 7, 91–102 (1999).

    Article  CAS  Google Scholar 

  39. Li, W., Dennis, C.A., Moore, G.R., James, R. & Kleanthous, C. Protein–protein interaction specificity of Im9 for the endonuclease toxin colicin E9 defined by homologue-scanning mutagenesis. J. Biol. Chem. 272, 22253–22258 (1997).

    Article  CAS  Google Scholar 

  40. Wallis, R. et al. Protein–protein interactions in colicin E9 Dnase–immunity protein complexes. Cognate and noncognate interactions that span the mM–fM affinity range. Biochemistry 34, 13751–13759 (1995).

    Article  Google Scholar 

  41. Kuwata, K. et al. Structural and kinetic characterisation of early folding events in β-lactoglobulin. Nature Struct. Biol. 8, 151–154 (2001).

    Article  CAS  Google Scholar 

  42. Grantcharova, V.P., Riddle, D.S., Santiago, J.V. & Baker, D. Important role of hydrogen bonds in the structurally polarized transition state for folding of the src SH3 domain. Nature Struct. Biol. 5, 714–720 (1998).

    Article  CAS  Google Scholar 

  43. Martinez, J.C. & Serrano, L. The folding transition state between SH3 domains is conformationally restricted and evolutionarily conserved. Nature Struct. Biol. 6, 1010–1016 (1999).

    Article  CAS  Google Scholar 

  44. Li, L., Mirny, L.A. & Shakhnovich, E.I. Kinetics, thermodynamics and evolution of non-native interactions in a protein folding nucleus. Nature Struct. Biol. 7, 336–342. (2000).

    Article  CAS  Google Scholar 

  45. Mirny, L. & Shakhnovich, E.I. Protein folding theory: from lattice to all-atom models. Annu. Rev. Biophys. Biomol. Struct. 30, 361–396 (2001)

    Article  Google Scholar 

  46. Kraulis, P.J. Molscript — a program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946–950 (1991).

    Article  Google Scholar 

  47. Merrit, E.A. & Bacon, D.J. Raster3D: Photorealistic molecular graphics. Methods Enzymol. 277, 505–524 (1997).

    Article  Google Scholar 

  48. Dennis, C.A. et al. A structural comparison of the colicin immunity proteins Im7 and Im9 gives new insights into the molecular determinants of immunity- protein specificity. Biochem. J. 333, 183–191 (1998).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We would like to thank A. Ashcroft for analyzing the proteins using mass spectrometry, K. Ainley for technical assistance and A. Berry for help making figures. We are also very grateful to D. Otzen, S. Fonseca and members of the Radford lab for advice and help with molecular biology, and to H. Roder for helpful discussions. We acknowledge the Wellcome Trust, the BBSRC and the University of Leeds for financial support. A.P.C. and S.E.R. are members of the Astbury Centre for Structural Molecular Biology, which is part of the North of England Structural Biology Centre and is funded by the BBSRC.

Author information

Authors and Affiliations

  1. School of Biochemistry and Molecular Biology, University of Leeds, Mount Preston Street, Leeds, LS2 9JT, UK

    Andrew P. Capaldi & Sheena E. Radford

  2. School of Biological Sciences, University of East Anglia, Norwich, NR4 7TJ, UK

    Colin Kleanthous

Authors
  1. Andrew P. Capaldi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Colin Kleanthous
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sheena E. Radford
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sheena E. Radford.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Capaldi, A., Kleanthous, C. & Radford, S. Im7 folding mechanism: misfolding on a path to the native state. Nat Struct Mol Biol 9, 209–216 (2002). https://doi.org/10.1038/nsb757

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsb757

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing