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Ultrarapid mixing experiments reveal that Im7 folds via an on-pathway intermediate

Nature Structural Biology volume 8, pages 68–72 (2001)Cite this article

Abstract

Many proteins populate partially organized structures during folding. Since these intermediates often accumulate within the dead time (2–5 ms) of conventional stopped-flow and quench-flow devices, it has been difficult to determine their role in the formation of the native state. Here we use a microcapillary mixing apparatus, with a time resolution of ∼150 μs, to directly follow the formation of an intermediate in the folding of a four-helix protein, Im7. Quantitative kinetic modeling of folding and unfolding data acquired over a wide range of urea concentrations demonstrate that this intermediate ensemble lies on a direct path from the unfolded to the native state.

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Figure 1: Possible three-state kinetic schemes for protein folding.
Figure 2: Fluorescence changes during the refolding of Im7.
Figure 3: On-pathway and off-pathway fits to Im7 folding kinetics.
Figure 4: Free energy diagram for Im7 folding/unfolding.

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Acknowledgements

The work was supported by grants from the National Institutes of Health (H.R.) and the Biotechnology and Biological Sciences Research Council (BBSRC) (A.P.C. and S.E.R.). We also acknowledge with thanks support from the Wellcome Trust as well as helpful discussions with N. Kad, N. Ferguson and S. Gorski. The Astbury Centre for Structural Molecular Biology is part of the North of England Structural Biology Centre, which is funded by the BBSRC.

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Author notes

  1. Andrew P. Capaldi, M. C. Ramachandra Shastry and Colin Kleanthous: These authors contributed equally to this work.

Authors and Affiliations

  1. School of Biochemistry and Molecular Biology and the Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT, UK

    Andrew P. Capaldi & Sheena E. Radford

  2. Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, 19111, Pennsylvania, USA

    M. C. Ramachandra Shastry & Heinrich Roder

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

    Colin Kleanthous

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  1. Andrew P. Capaldi
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  2. M. C. Ramachandra Shastry
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  3. Colin Kleanthous
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  4. Heinrich Roder
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  5. Sheena E. Radford
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Correspondence to Heinrich Roder or Sheena E. Radford.

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Capaldi, A., Shastry, M., Kleanthous, C. et al. Ultrarapid mixing experiments reveal that Im7 folds via an on-pathway intermediate. Nat Struct Mol Biol 8, 68–72 (2001). https://doi.org/10.1038/83074

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