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.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on SpringerLink
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout




Similar content being viewed by others
References
Dobson, C. M. Protein folding and misfolding. Nature 426, 884–890 (2003)
Fersht, A. R. & Daggett, V. Protein folding and unfolding at atomic resolution. Cell 108, 573–582 (2002)
Dyson, H. J. & Wright, P. E. Intrinsically unstructured proteins and their functions. Nature Rev. Mol. Cell Biol. 6, 197–208 (2005)
Chiti, F. & Dobson, C. M. Protein misfolding, functional amyloid, and human disease. Annu. Rev. Biochem. 75, 333–366 (2006)
Neidigh, J. W., Fesinmeyer, R. M. & Andersen, N. H. Designing a 20-residue protein. Nature Struct. Biol. 9, 425–430 (2002)
Kaptein, R. in Biological Magnetic Resonance (eds Berliner, L. J. & Reuben, J.) 145–191 (Plenum, New York, 1982)
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)
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)
Dyson, H. J. & Wright, P. E. Unfolded proteins and protein folding studied by NMR. Chem. Rev. 104, 3607–3622 (2004)
Klein-Seetharaman, J. et al. Long-range interactions within a nonnative protein. Science 295, 1719–1722 (2002)
Shortle, D. & Ackerman, M. S. Persistence of native-like topology in a denatured protein in 8 M urea. Science 293, 487–489 (2001)
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)
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)
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)
Balbach, J. et al. Detection of residue contacts in a protein folding intermediate. Proc. Natl Acad. Sci. USA 94, 7182–7185 (1997)
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)
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)
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)
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)
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)
Zhou, R. Trp-cage: Folding free energy landscape in explicit water. Proc. Natl Acad. Sci. USA 100, 13280–13285 (2003)
Mok, K. H. & Hore, P. J. Photo-CIDNP NMR methods for studying protein folding. Methods 34, 75–87 (2004)
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)
Kohn, J. E. et al. Random-coil behavior and the dimensions of chemically unfolded proteins. Proc. Natl Acad. Sci. USA 101, 12491–12496 (2004)
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)
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)
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)
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)
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)
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)
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.
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Competing interests
Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.
Supplementary information
Supplementary Information
This file contains Supplementary Methods, Supplementary Figures 1-5 with Legends, Supplementary Tables 1-2 and additional references (PDF 2424 kb)
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nature05728