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.

  • Letter
  • Published:

Folding dynamics of the B1 domain of protein G explored by ultrarapid mixing

Nature Structural Biology volume 6, pages 943–947 (1999)Cite this article

Abstract

For many proteins, compact conformations are known to accumulate in advance of the rate-limiting step in folding. To understand the nature and significance of these early conformational events, we employed ultrarapid mixing methods to fully characterize the kinetics of folding of the 57-residue B1 domain of protein G. Continuous-flow fluorescence measurements exhibit a major exponential phase on the submillisecond time scale (600–700 μs), which is followed by a slower phase with a denaturant-dependent time constant (2–30 ms) observable by conventional stopped-flow measurements. The combined kinetic traces quantitatively account for the total change in Trp 43 fluorescence upon folding, including the previously unresolved 'burst phase' signal. The denaturant dependence of the two rate constants and their relative amplitudes are fully consistent with a three-state mechanism, U ⇌ I ⇌ N, where I is a productive intermediate with native-like fluorescence properties. The relatively slow rate and exponential time course of the initial folding phase indicates that a substantial free energy barrier is encountered during chain condensation, resulting in a partially organized ensemble of states distinct from the initial unfolded conformations.

Access through your institution
Buy or subscribe

This is a preview of subscription content, access via your institution

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Tryptophan fluorescence changes during refolding of GB1 (20 mM) at 1.12 M GuHCl (0.4 M Na2SO4, 20 mM sodium acetate, pH 5), measured in matching continuous-flow (circles) and stopped-flow (diamonds) experiments at 20 °C, plotted on a logarithmic time scale (lower panel).
Figure 2: Effect of GuHCl concentration on the kinetics of refolding of GB1 in the presence of 0.4 M Na2SO4 (pH 5, 20 °C).
Figure 3: Variation of a, rates and b, amplitudes of the two kinetic phases involved in GB1 folding with GuHCl concentration.
Figure 4: Free energy diagrams under strongly and marginally stabilizing conditions predicted by the three-state analysis of the kinetic data in Fig. 3.

Similar content being viewed by others

References

  1. Gruenwald, B., Nicola, C.U., Lustig, A., Schwarz, G. & Klump, H. Biophys. Chem. 9, 137– 147 (1979).

    Article  Google Scholar 

  2. Williams, S. et al. Biochemistry 35, 691– 697 (1996).

    Article  CAS  Google Scholar 

  3. Muñoz, V., Thompson, P.A., Hofrichter, J. & Eaton, W.A. Nature 390, 196–199 ( 1997).

    Article  Google Scholar 

  4. Thompson, P.A., Eaton, W.A. & Hofrichter, J. Biochemistry 36, 9200– 9210 (1997).

    Article  CAS  Google Scholar 

  5. Gilmanshin, R., Williams, S., Callender, R.H., Woodruff, W.H. & Dyer, R.B. Proc. Natl. Acad. Sci. USA 94, 3709–3713 (1997).

    Article  CAS  Google Scholar 

  6. Eaton, W.A., Muñoz, V., Thompson, P.A., Henry, E.R. & Hofrichter, J. Accts. Chem. Res. 31, 745–753 (1998).

    Article  CAS  Google Scholar 

  7. Callender, R.H., Dyer, R.B., Gilmanshin, R. & Woodruff, W.H. Annu. Rev. Phys. Chem. 49, 173–202 (1998).

    Article  CAS  Google Scholar 

  8. Nölting, B., Golbik, R. & Fersht, A.R. Proc. Natl. Acad. Sci. USA 92, 10668–10672 (1995).

    Article  Google Scholar 

  9. Ballew, R.M., Sabelko, J. & Gruebele, M. Proc. Natl. Acad. Sci. USA 93, 5759–5764 (1996).

    Article  CAS  Google Scholar 

  10. Shastry, M.C.R. & Roder, H. Nature Struct. Biol. 5, 385–392 ( 1998).

    Article  CAS  Google Scholar 

  11. Jackson, S.E. Fold. Des. 3, R81–R91 ( 1998).

    Article  CAS  Google Scholar 

  12. Raschke, T.M. & Marqusee, S. Nature Struct. Biol. 4, 298–304 (1997).

    Article  CAS  Google Scholar 

  13. Nath, U. & Udgaonkar, J.B. Biochemistry 36, 8602–8610 (1997).

    Article  CAS  Google Scholar 

  14. Walkenhorst, W.F., Green, S.M. & Roder, H. Biochemistry 36, 5795– 5805 (1997).

    Article  CAS  Google Scholar 

  15. Heidary, D.K., Gross, L.A., Roy, M. & Jennings, P.A. Nature Struct. Biol. 4, 725–731 ( 1997).

    Article  CAS  Google Scholar 

  16. Parker, M.J., Lorch, M., Sessions, R.B. & Clarke, A.R. Biochemistry 37, 2538–2545 (1998).

    Article  CAS  Google Scholar 

  17. Capaldi, A.P., Ferguson, S.J. & Radford, S.E. J. Mol. Biol. 286, 1621– 1632 (1999).

    Article  CAS  Google Scholar 

  18. Roder, H. & Colón, W. Curr. Opin. Struct. Biol. 7, 15–28 (1997).

    Article  CAS  Google Scholar 

  19. Houry, W.A., Rothwarf, D.M. & Scheraga, H.A. Nature Struct. Biol. 2, 495 –503 (1995).

    Article  CAS  Google Scholar 

  20. Parker, M.J., Spencer, J. & Clarke, A.R. J. Mol. Biol. 253, 771– 786 (1995).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  22. Fujita, H. Polymer solutions (Elsevier, Amsterdam; 1990).

    Google Scholar 

  23. Dill, K.A. et al. Protein Sci. 4, 561– 602 (1995).

    Article  CAS  Google Scholar 

  24. Fersht, A.R. Proc. Natl. Acad. Sci. USA 92, 10869– 10873 (1995).

    Article  CAS  Google Scholar 

  25. Sosnick, T.R., Mayne, L. & Englander, S.W. Proteins 24, 413– 426 (1996).

    Article  CAS  Google Scholar 

  26. Creighton, T.E. Trends Biochem. Sci. 22, 6–11 (1997).

    Article  CAS  Google Scholar 

  27. Sosnick, T.R., Shtilerman, M.D., Mayne, L. & Englander, S.W. Proc. Natl. Acad. Sci. USA 94, 8545– 8550 (1997).

    Article  CAS  Google Scholar 

  28. Qi, P.X., Sosnick, T.R. & Englander, S.W. Nature Struct. Biol. 5, 882 –884 (1998).

    Article  CAS  Google Scholar 

  29. Regenfuss, P., Clegg, R.M., Fulwyler, M.J., Barrantes, F.J. & Jovin, T.M. Rev. Sci. Instrum. 56 , 283–290 (1985).

    Article  CAS  Google Scholar 

  30. Chan, C.-K. et al. Proc. Natl. Acad. Sci. USA 94, 1779 –1784 (1997).

    Article  CAS  Google Scholar 

  31. Shastry, M.C.R., Luck, S.D. & Roder, H. Biophys. J. 74, 2714– 2721 (1998).

    Article  CAS  Google Scholar 

  32. Gronenborn, A.M. et al. Science 253, 657–661 (1991).

    Article  CAS  Google Scholar 

  33. Achari, A. et al. Biochemistry 31, 10449– 10457 (1992).

    Article  CAS  Google Scholar 

  34. Kuszewski, J., Clore, G.M. & Gronenborn, A.M. Protein Sci. 3, 1945– 1952 (1994).

    Article  CAS  Google Scholar 

  35. Park, S.-H., O'Neil, K.T. & Roder, H. Biochemistry 36, 14277– 14283 (1997).

    Article  CAS  Google Scholar 

  36. Matouschek, A., Kellis, J.T.J., Serrano, L., Bycroft, M. & Fersht, A.R. Nature 346, 440–445 (1990).

    Article  CAS  Google Scholar 

  37. Houry, W.A., Rothwarf, D.M. & Scheraga, H.A. Biochemistry 33, 2516– 2530 (1994).

    Article  CAS  Google Scholar 

  38. Wolynes, P.G., Luthey-Schulten, Z. & Onuchic, J.N. Chem. Biol. 3, 425– 432 (1996).

    Article  CAS  Google Scholar 

  39. Hagen, S.J., Hofrichter, J., Szabo, A. & Eaton, W.A. Proc. Natl. Acad. Sci. USA 93, 11615– 11617 (1996).

    Article  CAS  Google Scholar 

  40. Jones, C.M. et al. Proc. Natl. Acad. Sci. USA 90, 11860 –11864 (1993).

    Article  CAS  Google Scholar 

  41. Shastry, M.C.R., Sauder, J.M. & Roder, H. Accts. Chem. Res. 31, 717– 725 (1998).

    Article  CAS  Google Scholar 

  42. Neri, D., Billeter, M., Wider, G. & Wüthrich, K. Science 257, 1559–1563 ( 1992).

    Article  CAS  Google Scholar 

  43. Tanford, C. Adv. Prot. Chem. 23, 121–282. (1968).

    CAS  Google Scholar 

  44. Bryngelson, J.D., Onuchic, J.N., Socci, N.D. & Wolynes, P.G. Proteins 21, 167–195 ( 1995).

    Article  CAS  Google Scholar 

  45. Plaxco, K.W., Simons, K.T. & Baker, D. J. Mol. Biol. 277, 985– 994 (1998).

    Article  CAS  Google Scholar 

  46. Blanco, F.J., Ortiz, A.R. & Serrano, L. Fold. Des. 2, 123– 133 (1997).

    Article  CAS  Google Scholar 

  47. Schindler, T., Herrler, M., Marahiel, M.A. & Schmid, F.X. Nature Struct. Biol. 2, 663–673 (1995).

    Article  CAS  Google Scholar 

  48. Burton, R.E., Huang, G.S., Daugherty, M.A., Fullbright, P.W. & Oas, T.G. J. Mol. Biol. 263, 311–322 (1996).

    Article  CAS  Google Scholar 

  49. Kiefhaber, T. Proc. Natl. Acad. Sci. USA 92, 9029– 9033 (1995).

    Article  CAS  Google Scholar 

  50. Baldwin, R.L. Fold. Des. 1, R1–R8 ( 1996).

    Article  CAS  Google Scholar 

  51. Matouschek, A., Serrano, L. & Fersht, A.R. J. Mol. Biol. 224, 819– 835 (1992).

    Article  CAS  Google Scholar 

  52. Parker, M.J., Dempsey, C.E., Hosszu, L.L.P., Waltho, J.P. & Clarke, A.R. Nature Struct. Biol. 5, 194–198 (1998).

    Article  CAS  Google Scholar 

  53. Dalby, P.A., Oliveberg, M. & Fersht, A.R. J. Mol. Biol. 276, 625– 646 (1998).

    Article  CAS  Google Scholar 

  54. Sheinerman, F.B. & Brooks, C.L.I. J. Mol. Biol. 278, 439–456 ( 1998).

    Article  CAS  Google Scholar 

  55. Alonso, D.O.V. & Daggett, V. Protein Sci. 7, 860–874 (1998).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank R.L. Dunbrack, H. Hensley and J.M. Sauder for helpful discussions and comments on the manuscript. This work was supported by a grant from the National Institutes of Health and an appropriation from the Commonwealth of Pennsylvania.

Author information

Authors and Affiliations

  1. Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, 19111, Pennsylvania, USA

    Soon-Ho Park, M.C. Ramachandra Shastry & Heinrich Roder

  2. Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, 19104, Pennsylvania, USA

    Heinrich Roder

Authors
  1. Soon-Ho Park
    View author publications

    Search author on:PubMed Google Scholar

  2. M.C. Ramachandra Shastry
    View author publications

    Search author on:PubMed Google Scholar

  3. Heinrich Roder
    View author publications

    Search author on:PubMed Google Scholar

Corresponding author

Correspondence to Heinrich Roder.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Park, SH., Shastry, M. & Roder, H. Folding dynamics of the B1 domain of protein G explored by ultrarapid mixing. Nat Struct Mol Biol 6, 943–947 (1999). https://doi.org/10.1038/13311

Download citation

  • Received:

  • Accepted:

  • Issue date:

  • DOI: https://doi.org/10.1038/13311

This article is cited by

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