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A pre-existing hydrophobic collapse in the unfolded state of an ultrafast folding protein

Nature volume 447, pages 106–109 (2007)Cite this article

Abstract

Insights into the conformational passage of a polypeptide chain across its free energy landscape have come from the judicious combination of experimental studies and computer simulations1,2. Even though some unfolded and partially folded proteins are now known to possess biological function3 or to be involved in aggregation phenomena associated with disease states1,4, experimentally derived atomic-level information on these structures remains sparse as a result of conformational heterogeneity and dynamics. Here we present a technique that can provide such information. Using a ‘Trp-cage’ miniprotein known as TC5b (ref. 5), we report photochemically induced dynamic nuclear polarization NMR6 pulse-labelling experiments that involve rapid in situ protein refolding7,8. These experiments allow dipolar cross-relaxation with hyperpolarized aromatic side chain nuclei in the unfolded state to be identified and quantified in the resulting folded-state spectrum. We find that there is residual structure due to hydrophobic collapse in the unfolded state of this small protein, with strong inter-residue contacts between side chains that are relatively distant from one another in the native state. Prior structuring, even with the formation of non-native rather than native contacts, may be a feature associated with fast folding events in proteins.

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Figure 1: The photo-CIDNP pulse labelling technique.
Figure 2: The 600 MHz 1 H NMR and photo-CIDNP spectra of TC5b.
Figure 3: The 600 MHz photo-CIDNP pulse-labelled NMR spectra of TC5b.
Figure 4: Representation of the three-dimensional structures of TC5b.

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References

  1. Dobson, C. M. Protein folding and misfolding. Nature 426, 884–890 (2003)

    Article  ADS  CAS  Google Scholar 

  2. Fersht, A. R. & Daggett, V. Protein folding and unfolding at atomic resolution. Cell 108, 573–582 (2002)

    Article  CAS  Google Scholar 

  3. Dyson, H. J. & Wright, P. E. Intrinsically unstructured proteins and their functions. Nature Rev. Mol. Cell Biol. 6, 197–208 (2005)

    Article  CAS  Google Scholar 

  4. Chiti, F. & Dobson, C. M. Protein misfolding, functional amyloid, and human disease. Annu. Rev. Biochem. 75, 333–366 (2006)

    Article  CAS  Google Scholar 

  5. Neidigh, J. W., Fesinmeyer, R. M. & Andersen, N. H. Designing a 20-residue protein. Nature Struct. Biol. 9, 425–430 (2002)

    Article  CAS  Google Scholar 

  6. Kaptein, R. in Biological Magnetic Resonance (eds Berliner, L. J. & Reuben, J.) 145–191 (Plenum, New York, 1982)

    Book  Google Scholar 

  7. Mok, K. H. et al. A rapid sample-mixing technique for transient NMR and photo-CIDNP spectroscopy: Applications to real-time protein folding. J. Am. Chem. Soc. 125, 12484–12492 (2003)

    Article  CAS  Google Scholar 

  8. Mok, K. H., Nagashima, T., Day, I. J., Hore, P. J. & Dobson, C. M. Multiple subsets of side-chain packing in partially folded states of α-lactalbumins. Proc. Natl Acad. Sci. USA 102, 8899–8904 (2005)

    Article  ADS  CAS  Google Scholar 

  9. Dyson, H. J. & Wright, P. E. Unfolded proteins and protein folding studied by NMR. Chem. Rev. 104, 3607–3622 (2004)

    Article  CAS  Google Scholar 

  10. Klein-Seetharaman, J. et al. Long-range interactions within a nonnative protein. Science 295, 1719–1722 (2002)

    Article  ADS  CAS  Google Scholar 

  11. Shortle, D. & Ackerman, M. S. Persistence of native-like topology in a denatured protein in 8 M urea. Science 293, 487–489 (2001)

    Article  CAS  Google Scholar 

  12. Yi, Q., Scalley-Kim, M. L., Alm, E. J. & Baker, D. NMR characterization of residual structure in the denatured state of protein L. J. Mol. Biol. 299, 1341–1351 (2000)

    Article  CAS  Google Scholar 

  13. Crowhurst, K. A. & Forman-Kay, J. D. Aromatic and methyl NOEs highlight hydrophobic clustering in the unfolded state of an SH3 domain. Biochemistry 42, 8687–8695 (2003)

    Article  CAS  Google Scholar 

  14. Religa, T. L., Markson, J. S., Mayor, U., Freund, S. M. V. & Fersht, A. R. Solution structure of a protein denatured state and folding intermediate. Nature 437, 1053–1056 (2005)

    Article  ADS  CAS  Google Scholar 

  15. Balbach, J. et al. Detection of residue contacts in a protein folding intermediate. Proc. Natl Acad. Sci. USA 94, 7182–7185 (1997)

    Article  ADS  CAS  Google Scholar 

  16. Qiu, L., Pabit, S. A., Roitberg, A. E. & Hagen, S. J. Smaller and faster: The 20-residue Trp-cage protein folds in 4 μs. J. Am. Chem. Soc. 124, 12952–12953 (2002)

    Article  CAS  Google Scholar 

  17. Simmerling, C., Strockbine, B. & Roitberg, A. E. All-atom structure prediction and folding simulations of a stable protein. J. Am. Chem. Soc. 124, 11258–11259 (2002)

    Article  CAS  Google Scholar 

  18. Snow, C. D., Zagrovic, B. & Pande, V. S. The Trp cage: Folding kinetics and unfolded state topology via molecular dynamics simulations. J. Am. Chem. Soc. 124, 14548–14549 (2002)

    Article  CAS  Google Scholar 

  19. Chowdhury, S., Lee, M. C., Xiong, G. & Duan, Y. Ab initio folding simulation of the Trp-cage mini-protein approaches NMR resolution. J. Mol. Biol. 327, 711–717 (2003)

    Article  CAS  Google Scholar 

  20. Pitera, J. W. & Swope, W. Understanding folding and design: Replica-exchange simulations of “Trp-cage” miniproteins. Proc. Natl Acad. Sci. USA 100, 7587–7592 (2003)

    Article  ADS  CAS  Google Scholar 

  21. Zhou, R. Trp-cage: Folding free energy landscape in explicit water. Proc. Natl Acad. Sci. USA 100, 13280–13285 (2003)

    Article  ADS  CAS  Google Scholar 

  22. Mok, K. H. & Hore, P. J. Photo-CIDNP NMR methods for studying protein folding. Methods 34, 75–87 (2004)

    Article  CAS  Google Scholar 

  23. Hore, P. J., Egmond, M. R., Edzes, H. T. & Kaptein, R. Cross-relaxation effects in the photo-CIDNP spectra of amino acids in proteins. J. Magn. Reson. 49, 122–150 (1982)

    ADS  CAS  Google Scholar 

  24. Kohn, J. E. et al. Random-coil behavior and the dimensions of chemically unfolded proteins. Proc. Natl Acad. Sci. USA 101, 12491–12496 (2004)

    Article  ADS  CAS  Google Scholar 

  25. Neuweiler, H., Doose, S. & Sauer, M. A microscopic view of miniprotein folding: Enhanced folding efficiency through formation of an intermediate. Proc. Natl Acad. Sci. USA 102, 16650–16655 (2005)

    Article  ADS  CAS  Google Scholar 

  26. Ahmed, Z., Beta, I. A., Mikhonin, A. V. & Asher, S. A. UV-resonance Raman thermal unfolding study of Trp-cage shows that it is not a simple two-state miniprotein. J. Am. Chem. Soc. 127, 10943–10950 (2005)

    Article  CAS  Google Scholar 

  27. Bunagan, M. R., Yang, X., Saven, J. G. & Gai, F. Ultrafast folding of a computationally designed Trp-cage mutant: Trp2-cage. J. Phys. Chem. B 110, 3759–3763 (2006)

    Article  CAS  Google Scholar 

  28. Sánchez, 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  Google Scholar 

  29. Khan, F., Kuprov, I., Craggs, T. D., Hore, P. J. & Jackson, S. E. 19F NMR studies of the native and denatured states of green fluorescent protein. J. Am. Chem. Soc. 128, 10729–10737 (2006)

    Article  CAS  Google Scholar 

  30. Lyon, C. E., Jones, J. A., Redfield, C., Dobson, C. M. & Hore, P. J. Two-dimensional 15N-1H photo-CIDNP as a surface probe of native and partially structured proteins. J. Am. Chem. Soc. 121, 6505–6506 (1999)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank I. Kuprov for the synthesis of 9-fluorenylmethoxycarbonyl-O-t-butyl-3-fluoro-l-Tyr and for NMR experiments on the 19F-Tyr-TC5b variant; T. Nagashima, C. J. V. Jones and H. Paisley for help with the conceptual design, building and testing of the rapid mixing injector; R. Gerber for assistance in acquiring the NMR spectra; A. L. Davis for discussions and for providing spectrometer time for the diffusion measurements; L. J. Smith, C. Redfield, A. E. Mark and D. A. C. Beck for discussions; and S. Min for assistance in figure preparation. K.H.M. also thanks M. Nilges, R. Wade and the EMBO Practical Course on Biomolecular Simulation. We are indebted to C. M. Dobson for continued encouragement in the application of photo-CIDNP to protein folding problems. This work was supported by the BBSRC (K.H.M., L.T.K., and P.J.H.), the Studienstiftung des deutschen Volkes (L.T.K.), the Deutsche Forschungsgemeinschaft (M.G.), and the US National Institutes of Health (N.H.A. and J.C.L.).

Author Contributions K.H.M. and I.J.D. built the in situ rapid mixing injector. K.H.M., M.G., I.J.D. and P.J.H. designed the experiments. K.H.M., L.T.K., M.G. and I.J.D. performed the experiments. J.C.L. and N.H.A. contributed the TC5b sample. M.G. developed the mathematical methods for obtaining NOE contact distances. K.H.M., L.T.K., M.G., I.J.D., N.H.A. and P.J.H. analysed the data. K.H.M., M.G., N.H.A. and P.J.H. wrote the paper. All authors discussed the results and commented on the manuscript.

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Author notes

  1. Iain J. Day

    Present address: Present address: Medway Sciences, University of Greenwich, Medway University Campus, Central Avenue, Chatham Maritime, Chatham, Kent, ME4 4TB, UK.,

Authors and Affiliations

  1. Department of Chemistry, University of Oxford, Physical & Theoretical Chemistry Laboratory, South Parks Road, Oxford OX1 3QZ, UK,

    K. Hun Mok, Lars T. Kuhn & P. J. Hore

  2. School of Biochemistry and Immunology, Trinity College, University of Dublin, Dublin 2, Ireland,

    K. Hun Mok

  3. Fachbereich Chemie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Strasse 2, D-06120 Halle/Saale, Germany,

    Martin Goez

  4. Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, Massachusetts 01605, USA,

    Iain J. Day

  5. Department of Chemistry, University of Washington, Seattle, Washington 98195, USA,

    Jasper C. Lin & Niels H. Andersen

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This file contains Supplementary Methods, Supplementary Figures 1-5 with Legends, Supplementary Tables 1-2 and additional references (PDF 2424 kb)

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Mok, K., Kuhn, L., Goez, M. et al. A pre-existing hydrophobic collapse in the unfolded state of an ultrafast folding protein. Nature 447, 106–109 (2007). https://doi.org/10.1038/nature05728

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