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Kinetics of unfolding and folding from amide hydrogen exchange in native ubiquitin

Nature Structural Biology volume 8, pages 331–333 (2001)Cite this article

Abstract

Amide hydrogen (NH) exchange is one of the few experimental techniques with the potential for determining the thermodynamics and kinetics of conformational motions at nearly every residue in native proteins. Quantitative interpretation of NH exchange in terms of molecular motions relies on a simple two-state kinetic model: at any given slowly exchanging NH, a closed or exchange-incompetent conformation is in equilibrium with an open or exchange-competent conformation. Previous studies have demonstrated the accuracy of this model in measuring conformational equilibria by comparing exchange data with the thermodynamics of protein unfolding. We report here a test of the accuracy of the model in determining the kinetics of conformational changes in native proteins. The kinetics of folding and unfolding for ubiquitin have been measured by conventional methods and compared with those derived from a comprehensive analysis of the pH dependence of exchange in native ubiquitin. Rate constants for folding and unfolding from these two very different types of experiments show good agreement. The simple model for NH exchange thus appears to be a robust framework for obtaining quantitative information about molecular motions in native proteins.

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Figure 1: Comparison of folding kinetics in ubiquitin as measured by saturation transfer and NH exchange.
Figure 2: The pH dependence of exchange for the most slowly exchanging NHs in native ubiquitin in the presence of 1.5 M GdnDCl.

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References

  1. Englander, S.W. Sosnick, T.R., Englander, J.J. & Mayne, L. Curr. Opin. Struct. Biol. 6, 18–23 (1996).

    Article  CAS  Google Scholar 

  2. Li, R. & Woodward, C. Protein Sci. 8, 1571–1590 (1999).

    Article  CAS  Google Scholar 

  3. Bai, Y., Milne, J.S., Mayne, L. & Englander, S. W. Proteins 17, 75–86 (1993).

    Article  CAS  Google Scholar 

  4. Mayo, S. L. & Baldwin, R. L. Science 262, 873–876 (1993).

    Article  CAS  Google Scholar 

  5. Kim, K. S., Fuchs, J. A. & Woodward, C. K. Biochemistry 32, 9600–9608 (1993).

    Article  CAS  Google Scholar 

  6. Perrett, S., Clarke, J., Hounslow, A. M. & Fersht, A. R. Biochemistry 34, 9288–9298 (1995).

    Article  CAS  Google Scholar 

  7. Bai, Y., Sosnick, T. R., Mayne, L. & Englander, S. W. Science 269, 192–197 (1995).

    Article  CAS  Google Scholar 

  8. Huyghues-Despointes, B. M. P., Scholtz, J. M. & Pace, C. N. Nature Struct. Biol. 6, 910–912 (1999).

    Article  CAS  Google Scholar 

  9. Tanford, C. Adv. Protein Chem. 24, 1–95 (1970).

    Article  CAS  Google Scholar 

  10. Roder, H., Wagner, G. & Wüthrich, K. Biochemistry 24, 7396–7407 (1985).

    Article  CAS  Google Scholar 

  11. Arrington, C.B. & Robertson, A.D. Biochemistry 36, 8686–8691 (1997).

    Article  CAS  Google Scholar 

  12. Parker, M.J., Dempsey, C.E., Hosszu, L.L. P., Waltho, J.P. & Clarke, A.R. Nature Struct. Biol. 5, 194–197 (1998).

    Article  CAS  Google Scholar 

  13. Kragelund, B.B., Heinemann, B., Knudsen, J. & Poulsen, F.M. Protein Sci. 7, 2237–2248 (1998)

    Article  CAS  Google Scholar 

  14. Arrington, C.B. & Robertson, A.D. J. Mol. Biol. 296, 1307–1317 (2000).

    Article  CAS  Google Scholar 

  15. Clarke, J. & Fersht, A.R. Folding Des. 1, 243–254 (1996).

    Article  CAS  Google Scholar 

  16. Briggs, M. S. & Roder, H. Proc. Natl. Acad. Sci. USA 89, 2017–2021 (1992).

    Article  CAS  Google Scholar 

  17. Gladwin, S.T. & Evans, P.A. Folding Des. 1, 407–417 (1996).

    Article  CAS  Google Scholar 

  18. Johnson, E.C., Lazar, G.A., Desjarlasis, J.R. & Handel, T.M. Structure 7, 967–976 (1999).

    Article  CAS  Google Scholar 

  19. Dobson, C.M. & Evans, P.A. Biochemistry 23, 4267–4270 (1984).

    Article  CAS  Google Scholar 

  20. Roder, H. Methods Enzymol. 176, 446–473 (1989).

    Article  CAS  Google Scholar 

  21. Khorasanizadeh, S., Peters, I.D. & Roder, H. Nature Struct. Biol. 3, 193–205 (1996).

    Article  CAS  Google Scholar 

  22. Krantz, B.A. & Sosnick, T.R. Biochemistry 39, 11696–11701 (2000).

    Article  CAS  Google Scholar 

  23. Arrington, C.B., Teesch, L.M. & Robertson, A.D. J. Mol. Biol. 285, 1265–1275 (1999).

    Article  CAS  Google Scholar 

  24. Arrington, C.B. & Robertson, A.D. J. Mol. Biol. 300, 221–232 (2000).

    Article  CAS  Google Scholar 

  25. Fersht, A. Structure and Mechanism in Protein Science (W. H. Freeman and Co., New York; 1999).

    Google Scholar 

  26. Pace, C.N. Methods Enzymol. 131, 266–280 (1986).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank J. Elwood for technical assistance and R. Cohen for careful reading of the manuscript.

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Authors and Affiliations

  1. Department of Biochemistry, University of Iowa, Iowa City, 52242, Iowa, USA

    Thirunavukkarasu Sivaraman, Cammon B. Arrington & Andrew D. Robertson

  2. Medical Scientist Training Program, University of Iowa, Iowa City, 52242, Iowa, USA

    Cammon B. Arrington

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  1. Thirunavukkarasu Sivaraman
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  2. Cammon B. Arrington
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Correspondence to Andrew D. Robertson.

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Sivaraman, T., Arrington, C. & Robertson, A. Kinetics of unfolding and folding from amide hydrogen exchange in native ubiquitin. Nat Struct Mol Biol 8, 331–333 (2001). https://doi.org/10.1038/86208

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