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The mechanism of folding of Im7 reveals competition between functional and kinetic evolutionary constraints

Nature Structural & Molecular Biology volume 16, pages 318–324 (2009)Cite this article

Abstract

Many proteins reach their native state through pathways involving the presence of folding intermediates. It is not clear whether this type of folding landscape results from insufficient evolutionary pressure to optimize folding efficiency, or arises from a conflict between functional and folding constraints. Here, using protein-engineering, ultra-rapid mixing and stopped-flow experiments combined with restrained molecular dynamics simulations, we characterize the transition state for the formation of the intermediate populated during the folding of the bacterial immunity protein, Im7, and the subsequent molecular steps leading to the native state. The results provide a comprehensive view of the folding process of this small protein. An analysis of the contributions of native and non-native interactions at different stages of folding reveals how the complexity of the folding landscape arises from concomitant evolutionary pressures for function and folding efficiency.

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Figure 1: Native structure of Im7 and representative kinetic traces.
Figure 2: Dependence of the natural logarithm of the observed rate constants for folding/unfolding on the concentration of urea for selected Im7 variants: I7V (a), L19A (b), L38A (c), I54V (d), I72V (e) and A77G (f).
Figure 3: Calculated Φ-values.
Figure 4: Comparison of structural properties of TS1, I and TS2.
Figure 5: Schematic illustration of the folding landscape of Im7.
Figure 6: Non-nativeness during folding versus functionality.
Figure 7: Interaction patterns of selected residues in TS1, I, TS2 and N.

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Acknowledgements

We thank C. Kleanthous and members of the Radford group for helpful discussions, S. Masca and I. Rodriguez-Mendieta for much help with the design and construction of the ultra-rapid mixing device and C. Gell for help with data analysis. C.T.F. was supported by the UK Biotechnology and Biological Sciences Research Council BBSRC (24/B17145), M.V. by the European Molecular Biology Organization, the Leverhulme Trust and the Royal Society, and J.G. by the UK Medical Research Council.

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Author notes

  1. Claire T Friel & D Alastair Smith

    Present address: Present addresses: Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany (C.T.F.).,

  2. Avacta Group Plc, York Biocentre, Innovation Way, York YO10 5NY, UK (D.A.S.).

Authors and Affiliations

  1. Astbury Centre for Structural Molecular Biology, University of Leeds, Mount Preston Street, Leeds, LS2 9JT, UK

    Claire T Friel, D Alastair Smith & Sheena E Radford

  2. Institute of Molecular and Cellular Biology, University of Leeds, Mount Preston Street, Leeds, LS2 9JT, UK

    Claire T Friel & Sheena E Radford

  3. School of Physics and Astronomy, University of Leeds, Leeds, LS2 9JT, UK

    D Alastair Smith

  4. Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB1 1EW, UK

    Michele Vendruscolo

  5. Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, UK

    Joerg Gsponer

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Contributions

C.T.F. performed all experiments and their analysis and interpretation, and manuscript preparation; D.A.S. performed instrument development and analysis; M.V. performed molecular dynamics data interpretation and manuscript preparation; J.G. performed all molecular dynamics simulations and their analysis, interpretation and manuscript preparation; S.E.R. conceived of the project and prepared the manuscript.

Corresponding authors

Correspondence to Joerg Gsponer or Sheena E Radford.

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Supplementary Figures 1–5, Supplementary Tables 1 and 2 and Supplementary Methods (PDF 519 kb)

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Friel, C., Alastair Smith, D., Vendruscolo, M. et al. The mechanism of folding of Im7 reveals competition between functional and kinetic evolutionary constraints. Nat Struct Mol Biol 16, 318–324 (2009). https://doi.org/10.1038/nsmb.1562

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