SpringerLink
Log in

Low-populated folding intermediates of Fyn SH3 characterized by relaxation dispersion NMR

  • Letter
  • Published:

From Nature

View current issue Submit your manuscript

Abstract

Many biochemical processes proceed through the formation of functionally significant intermediates1,2. Although the identification and characterization of such species can provide vital clues about the mechanisms of the reactions involved, it is challenging to obtain information of this type in cases where the intermediates are transient or present only at low population1,2,3,4. One important example of such a situation involves the folding behaviour of small proteins that represents a model for the acquisition of functional structure in biology1. Here we use relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy to identify, for two mutational variants of one such protein, the SH3 domain from Fyn tyrosine kinase5, a low-population folding intermediate in equilibrium with its unfolded and fully folded states. By performing the NMR experiments at different temperatures, this approach has enabled characterization of the kinetics and energetics of the folding process as well as providing structures of the intermediates. A general strategy emerges for an experimental determination of the energy landscape of a protein by applying this methodology to a series of mutants whose intermediates have differing degrees of native-like structure.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price includes VAT (Canada)

Instant access to the full article PDF.

Institutional subscriptions

Figure 1: Measurement of kf and ku in the G48M and G48V Fyn SH3 domains.
Figure 2: Three-site folding model for G48M and G48V Fyn SH3.
Figure 3: Structural analysis of the I state of mutants G48M and G48V of Fyn SH3 using chemical shifts.

Similar content being viewed by others

References

  1. Fersht, A. Structure and Mechanism in Protein Science (W.H. Freeman and Company, New York, 1999)

    Google Scholar 

  2. Kahn, F., Chuang, J. I., Gianni, S. & Fersht, A. R. The kinetic pathway of folding of barnase. J. Mol. Biol. 333, 169–186 (2003)

    Article  Google Scholar 

  3. Sanchez, I. E. & Keifhaber, 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  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  5. Pawson, T. & Gish, G. D. SH2 and SH3 domains: from structure to function. Cell 71, 359–362 (1992)

    Article  CAS  Google Scholar 

  6. Jackson, S. E. How do small, single domain proteins fold? Fold. Design 3, R81–R91 (1998)

    Article  CAS  Google Scholar 

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

    ADS  CAS  Google Scholar 

  8. Daggett, V. & Fersht, A. R. The present view of the mechanism of protein folding. Nature Rev. Mol. Cell Biol. 4, 497–502 (2003)

    Article  CAS  Google Scholar 

  9. Northey, J. G. B., Di Nardo, A. A. & Davidson, A. R. Hydrophobic core packing in the SH3 domain folding transition state. Nature Struct. Biol. 9, 126–130 (2002)

    Article  CAS  Google Scholar 

  10. Plaxco, K. W. et al. The folding kinetics and thermodynamics of the Fyn-SH3 domain. Biochemistry 37, 2529–2537 (1998)

    Article  CAS  Google Scholar 

  11. Martinez, J. C. & Serrano, L. The folding transition state between SH3 domains is conformationally restricted and evolutionarily conserved. Nature Struct. Biol. 6, 1010–1016 (1999)

    Article  CAS  Google Scholar 

  12. Grantcharova, V. P., Riddle, D. S. & Baker, D. Long-range order in the src SH3 folding transition state. Proc. Natl Acad. Sci. USA 97, 7084–7089 (2000)

    Article  ADS  CAS  Google Scholar 

  13. Di Nardo, A. A., Korzhnev, D. M., Stogios, P. J., Zarrine-Afsar, A., Kay, L. E. & Davidson, A. R. Dramatic acceleration of protein folding by stabilization of a non-native backbone conformation. Proc. Natl Acad. Sci. USA 101, 7954–7959 (2004)

    Article  ADS  CAS  Google Scholar 

  14. Palmer, A. G., Kroenke, C. D. & Loria, J. P. NMR methods for quantifying microsecond-to-millisecond motions in biological macromolecules. Methods Enzymol. 339, 204–238 (2001)

    Article  CAS  Google Scholar 

  15. Mulder, F. A. A., Mittermaier, A., Hon, B., Dahlquist, F. W. & Kay, L. E. Studying excited states of protein by NMR spectroscopy. Nature Struct. Biol. 8, 932–935 (2001)

    Article  CAS  Google Scholar 

  16. Dunitz, J. D. Win some, lose some. Enthalpy–entropy compensation in weak intermolecular interactions. Chem. Biol. 2, 709–712 (1995)

    Article  CAS  Google Scholar 

  17. Oliveberg, M., Tan, Y. J. & Fersht, A. R. Negative activation enthalpies on the kinetics of protein folding. Proc. Natl Acad. Sci. USA 92, 8926–8929 (1995)

    Article  ADS  CAS  Google Scholar 

  18. Dobson, C. M., Sali, A. & Karplus, M. Protein folding: A perspective from theory and experiment. Angew. Chem. Int. Edn Engl. 37, 867–893 (1998)

    Article  Google Scholar 

  19. Vendruscolo, M., Paci, E., Dobson, C. M. & Karplus, M. Three key residues from a critical contact network in a protein folding transition state. Nature 409, 641–645 (2001)

    Article  ADS  CAS  Google Scholar 

  20. Xu, X. P. & Case, D. A. Probing multiple effects on 15N, 13Cα, 13Cβ, 13C′ chemical shifts in peptides using density functional theory. Biopolymers 65, 408–423 (2002)

    Article  CAS  Google Scholar 

  21. Northey, J. G. B., Maxwell, K. L. & Davidson, A. R. Probing folding kinetics beyond the Phi value: Using multiple amino acid substitutions to investigate the structure of the SH3 domain folding transition state. J. Mol. Biol. 320, 389–402 (2002)

    Article  CAS  Google Scholar 

  22. Maxwell, K. L. & Davidson, A. R. Mutagenesis of a buried polar interaction in an SH3 domain: sequence conservation provides the best prediction of stability effects. Biochemistry 37, 16172–16182 (1998)

    Article  CAS  Google Scholar 

  23. Bax, A. Multidimensional nuclear magnetic resonance methods for protein studies. Curr. Opin. Struct. Biol. 4, 738–744 (1994)

    Article  CAS  Google Scholar 

  24. Wishart, D. S. & Sykes, B. D. The 13C chemical-shift index: a simple method for the identification of protein secondary structure using 13C chemical-shift data. J. Biomol. NMR 4, 171–180 (1994)

    Article  CAS  Google Scholar 

  25. Tollinger, M., Skrynnikov, N. R., Mulder, F. A. A., Forman-Kay, J. D. & Kay, L. E. Slow dynamics in folded and unfolded states of an SH3 domain. J. Am. Chem. Soc. 123, 11341–11352 (2001)

    Article  CAS  Google Scholar 

  26. Paci, E., Vendruscolo, M., Dobson, C. M. & Karplus, M. Determination of a transition state at atomic resolution from protein engineering data. J. Mol. Biol. 324, 151–163 (2002)

    Article  CAS  Google Scholar 

  27. Noble, M. E., Musacchio, A., Saraste, M., Courtneidge, S. A. & Wierenga, R. K. Crystal structure of the SH3 domain in human Fyn; comparison of the three-dimensional structures of SH3 domains in tyrosine kinases and spectrin. EMBO J. 12, 2617–2624 (1993)

    Article  CAS  Google Scholar 

  28. Press, W. H., Flannery, B. P., Teukolsky, S. A. & Vetterling, W. T. Numerical Recipes in C (Cambridge Univ. Press, 1988)

    MATH  Google Scholar 

Download references

Acknowledgements

This work was supported through grants from the Canadian Institutes of Health Research (A.R.D, L.E.K and D.M.K.), the European Comission (X.S.), the Royal Society (M.V.), the Leverhulme Trust (M.V. and C.M.D.) and the Wellcome Trust (C.M.D.). L.E.K. holds a Canada Research Chair in Biochemistry.

Author information

Author notes

  1. Dmitry M. Korzhnev and Xavier Salvatella: These authors contributed equally to this work

Authors and Affiliations

  1. Departments of Molecular and Medical Genetics and Biochemistry, University of Toronto, Toronto, Ontario, M5S 1A8, Canada

    Dmitry M. Korzhnev, Ariel A. Di Nardo, Alan R. Davidson & Lewis E. Kay

  2. Department of Chemistry and Protein Engineering Network Center of Excellence, University of Toronto, Toronto, Ontario, M5S 1A8, Canada

    Dmitry M. Korzhnev & Lewis E. Kay

  3. Department of Chemistry, University of Cambridge, Cambridge, Lensfield Road, CB2 1EW, UK

    Xavier Salvatella, Michele Vendruscolo & Christopher M. Dobson

Authors
  1. Dmitry M. Korzhnev
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xavier Salvatella
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michele Vendruscolo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ariel A. Di Nardo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Alan R. Davidson
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Christopher M. Dobson
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Lewis E. Kay
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Christopher M. Dobson or Lewis E. Kay.

Ethics declarations

Competing interests

The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Information

Includes information on fitting of dispersion profiles and structure calculations; Table 1S. Folding kinetics parameters obtained from a global fit of CPMG dispersion data for Fyn SH3 mutants; Table 2S. X2 target functions obtained in global fits of CPMG dispersion data for 23(26) residues of G48M(G48V) measured at 5(4) temperatures and 3(2) magnetic fields using 2- and 3-site exchange models; Table 3S. Thermodynamic parameters for G48M(G48V) mutants of the Fyn SH3 domain obtained from a global fit of CPMG dispersion data using 3- and 2-site exchange models. Table 4S. Backbone 15N chemical shift differences between states F and I (δFI) and between states F and U (δFU) and their ratio Δexp=δFI/δFU for G48M and G48V mutants of the Fyn SH3 domain; Figure 1S. Average r.m.s deviation from the native state as a function of residue number for the calculated I states of G48M and G48V; Figure 2S. Relaxation dispersion curves recorded for G48M at concentrations of 1.0 and 0.3 mM, 15 and 30oC, do not depend on protein concentration. (PDF 282 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Korzhnev, D., Salvatella, X., Vendruscolo, M. et al. Low-populated folding intermediates of Fyn SH3 characterized by relaxation dispersion NMR. Nature 430, 586–590 (2004). https://doi.org/10.1038/nature02655

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature02655

  • Springer Nature Limited

This article is cited by

Search

Navigation