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.

Probing the free-energy surface for protein folding with single-molecule fluorescence spectroscopy

Nature volume 419, pages 743–747 (2002)Cite this article

A Corrigendum to this article was published on 02 January 2003

Abstract

Protein folding is inherently a heterogeneous process because of the very large number of microscopic pathways that connect the myriad unfolded conformations to the unique conformation of the native structure. In a first step towards the long-range goal of describing the distribution of pathways experimentally, Förster resonance energy transfer1 (FRET) has been measured on single, freely diffusing molecules2,3,4. Here we use this method to determine properties of the free-energy surface for folding that have not been obtained from ensemble experiments. We show that single-molecule FRET measurements of a small cold-shock protein expose equilibrium collapse of the unfolded polypeptide and allow us to calculate limits on the polypeptide reconfiguration time. From these results, limits on the height of the free-energy barrier to folding are obtained that are consistent with a simple statistical mechanical model, but not with the barriers derived from simulations using molecular dynamics. Unlike the activation energy, the free-energy barrier includes the activation entropy and thus has been elusive to experimental determination for any kinetic process in solution.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

Buy

All prices are NET prices.

Figure 1: Schematic structures of protein and polyproline helices labelled with donor (Alexa 488) and acceptor (Alexa 594) dyes (using the program MOL/MOL).
Figure 2: FRET trajectories and histograms.
Figure 3: Dependence of the means and widths of the measured FRET efficiency (Eapp) on the concentration of GdmCl.
Figure 4: Two limiting cases for polypeptide dynamics in experiments on freely diffusing molecules.

References

  1. Van Der Meer, B. W., Coker, G. III & Chen, S. S.-Y. Resonance Energy Transfer. Theory and Data (VHC, New York, 1994)

    Google Scholar 

  2. Jia, Y. W. et al. Folding dynamics of single GCN4 peptides by fluorescence energy transfer confocal microscopy. Chem. Phys. 247, 69–83 (1999)

    CAS  Article  Google Scholar 

  3. Talaga, D. S. et al. Dynamics and folding of single two-stranded coiled-coil peptides studied by fluorescent energy transfer confocal microscopy. Proc. Natl Acad. Sci. USA 97, 13021–13026 (2000)

    ADS  CAS  Article  Google Scholar 

  4. Deniz, A. A. et al. Single-molecule protein folding: diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2. Proc. Natl Acad. Sci. USA 97, 5179–5184 (2000)

    ADS  CAS  Article  Google Scholar 

  5. Perl, D. et al. Conservation of rapid two-state folding in mesophilic, thermophilic and hyperthermophilic cold shock proteins. Nature Struct. Biol. 5, 229–235 (1998)

    CAS  Article  Google Scholar 

  6. Mandelkern, L. in Poly-α-amino Acids. Protein Models for Conformational Studies (ed. Fasman, G. D.) 675–724 (Marcel Dekker, New York, 1967)

    Google Scholar 

  7. Jacob, J., Baker, B., Bryant, R. G. & Cafiso, D. S. Distance estimates from paramagnetic enhancements of nuclear relaxation in linear and flexible model peptides. Biophys. J. 77, 1086–1092 (1999)

    CAS  Article  Google Scholar 

  8. Stryer, L. & Haugland, R. P. Energy transfer: a spectroscopic ruler. Proc. Natl Acad. Sci. USA 58, 719–726 (1967)

    ADS  CAS  Article  Google Scholar 

  9. Wassenberg, D., Welker, C. & Jaenicke, R. Thermodynamics of the unfolding of the cold-shock protein from Thermotoga maritima. J. Mol. Biol. 289, 187–193 (1999)

    CAS  Article  Google Scholar 

  10. Fersht, A. Structure and Mechanism in Protein Science: a Guide to Enzyme Catalysis and Protein Folding (W. H. Freeman, New York, 1999)

    Google Scholar 

  11. Alonso, D. O. V. & Dill, K. A. Solvent denaturation and stabilization of globular proteins. Biochemistry 30, 5974–5985 (1991)

    CAS  Article  Google Scholar 

  12. Chan, C. K. et al. Submillisecond protein folding kinetics studied by ultrarapid mixing. Proc. Natl Acad. Sci. USA 94, 1779–1784 (1997)

    ADS  CAS  Article  Google Scholar 

  13. Millett, I. S., Doniach, S. & Plaxco, K. W. Towards a taxonomy of the denatured state: small angle scattering studies of unfolded proteins. Adv. Prot. Chem. (in the press)

  14. Allen, M. P. & Tildesley, D. J. Computer Simulation of Liquids 192 (Clarendon, Oxford, 1987)

    MATH  Google Scholar 

  15. Dahan, M. et al. Ratiometric measurement and identification of single diffusing molecules. Chem. Phys. 247, 85–106 (1999)

    CAS  Article  Google Scholar 

  16. Deniz, A. A. et al. Single-pair fluorescence resonance energy transfer on freely diffusing molecules: observation of Förster distance dependence and subpopulations. Proc. Natl Acad. Sci. USA 96, 3670–3675 (1999)

    ADS  CAS  Article  Google Scholar 

  17. Bryngelson, J. D. & Wolynes, P. G. Spin glasses and the statistical mechanics of protein folding. Proc. Natl Acad. Sci. USA 84, 7524–7528 (1987)

    ADS  CAS  Article  Google Scholar 

  18. Bryngelson, J. D., Onuchic, J. N., Socci, N. D. & Wolynes, P. G. Funnels, pathways, and the energy landscape of protein folding: a synthesis. Proteins Struct. Funct. Genet. 21, 167–195 (1995)

    CAS  Article  Google Scholar 

  19. Dobson, C. M., Sali, A. & Karplus, M. Protein folding: a perspective from theory and experiment. Angew. Chem. Int. Edit. 37, 868–893 (1998)

    Article  Google Scholar 

  20. Socci, N. D., Onuchic, J. N. & Wolynes, P. G. Diffusive dynamics of the reaction coordinate for protein folding funnels. J. Chem. Phys. 104, 5860–5868 (1996)

    ADS  CAS  Article  Google Scholar 

  21. Klimov, D. K. & Thirumalai, D. Viscosity dependence of the folding rates of proteins. Phys. Rev. Lett. 79, 317–320 (1997)

    ADS  CAS  Article  Google Scholar 

  22. Nymeyer, H., Socci, N. D. & Onuchic, J. N. Landscape approaches for determining the ensemble of folding transition states: Success and failure hinge on the degree of frustration. Proc. Natl Acad. Sci. USA 97, 634–639 (2000)

    ADS  CAS  Article  Google Scholar 

  23. Kramers, H. A. Brownian motion in a field of force and the diffusion model of chemical reactions. Physica 7, 284–304 (1940)

    ADS  MathSciNet  CAS  Article  Google Scholar 

  24. Lapidus, L. J., Steinbach, P. J., Eaton, W. A., Szabo, A. & Hofrichter, J. Effects of chain stiffness on the dynamics of loop formation in polypeptides. J. Phys. Chem. B (in the press)

  25. Jäger, M., Nguyen, H., Crane, J. C., Kelley, J. W. & Gruebele, M. The folding mechanism of a β-sheet: the WW domain. J. Mol. Biol. 311, 373–393 (2001)

    Article  Google Scholar 

  26. Hagen, S. J., Hofrichter, J. & Eaton, W. A. The rate of intrachain diffusion of unfolded cytochrome c. J. Phys. Chem. B 101, 2352–2365 (1997)

    CAS  Article  Google Scholar 

  27. Portman, J. J., Takada, S. & Wolynes, P. G. Microscopic theory of protein folding rates. II. Local reaction coordinates and chain dynamics. J. Chem. Phys. 114, 5082–5096 (2001)

    ADS  CAS  Article  Google Scholar 

  28. Muñoz, V. & Eaton, W. A. A simple model for calculating the kinetics of protein folding from three-dimensional structure. Proc. Natl Acad. Sci. USA 96, 11311–11316 (1999)

    ADS  Article  Google Scholar 

  29. Shea, J. E. & Brooks, C. L. From folding theories to folding proteins: A review and assessment of simulation studies of protein folding and unfolding. Annu. Rev. Phys. Chem. 52, 499–535 (2001)

    ADS  CAS  Article  Google Scholar 

  30. Reid, K. L., Rodriguez, H. M., Hillier, B. J. & Gregoret, L. M. Stability and folding properties of a model β-sheet protein, Escherichia coli CspA. Protein Sci. 7, 470–479 (1998)

    CAS  Article  Google Scholar 

  31. Kremer, W. et al. Solution NMR structure of the cold-shock protein from the hyperthermophilic bacterium Thermotoga maritima. Eur. J. Biochem. 268, 2527–2539 (2001)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank A. Szabo and I. Gopich for suggestions and guidance on theoretical issues; S. Doniach, J. Hofrichter and G. Hummer for discussion and comments on the manuscript; L. Davis and R. Zare for advice regarding the single-molecule instrument; and L. Pannell for mass spectroscopy measurements. B.S. was supported by an Emmy Noether fellowship from the Deutsche Forschungsgemeinschaft.

Author information

Author notes

  1. Benjamin Schuler and Everett A. Lipman: These authors contributed equally to this work

Authors and Affiliations

  1. Laboratory of Chemical Physics, Building 5, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, 20892-0520, USA

    Benjamin Schuler, Everett A. Lipman & William A. Eaton

Authors
  1. Benjamin Schuler
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Everett A. Lipman
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. William A. Eaton
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to William A. Eaton.

Ethics declarations

Competing interests

The authors declare that they have no competing financial interests.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Schuler, B., Lipman, E. & Eaton, W. Probing the free-energy surface for protein folding with single-molecule fluorescence spectroscopy. Nature 419, 743–747 (2002). https://doi.org/10.1038/nature01060

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature01060

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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