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Reaching the protein folding speed limit with large, sub-microsecond pressure jumps

Nature Methods volume 6, pages 515–519 (2009)Cite this article

Abstract

Biomolecules are highly pressure-sensitive, but their dynamics upon return to ambient pressure are often too fast to observe with existing approaches. We describe a sample-efficient method capable of large and very fast pressure drops (<1 nanomole, >2,500 atmospheres and <0.7 microseconds). We validated the method by fluorescence-detected refolding of a genetically engineered lambda repressor mutant from its pressure-denatured state. We resolved barrierless structure formation upon return to ambient pressure; we observed a 2.1 ± 0.7 microsecond refolding time, which is very close to the 'speed limit' for proteins and much faster than the corresponding temperature-jump refolding of the same protein. The ability to experimentally perform a large and very fast pressure drop opens up a new region of the biomolecular energy landscape for atomic-level simulation.

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Figure 1: Pressure-drop apparatus.
Figure 2: Schematic timing diagram and raw experimental data.
Figure 3: Pressure denaturation of λ*YG analyzed by singular value decomposition (SVD).
Figure 4: Pressure jumps of λ*YG from 250 MPa to 0.1 MPa.
Figure 5: Single-shot tryptophan instrument response.

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References

  1. Hartl, F.U. Molecular chaperones in cellular protein folding. Nature 381, 571–580 (1996).

    Article  CAS  Google Scholar 

  2. Martin, J.S., Brown, L. & Haberstroh, K. Microfilaments are involved in renal cell responses to sustained hydrostatic pressure. J. Urol. 173, 1410–1417 (2005).

    Article  Google Scholar 

  3. Eigen, M. & Maeyer, L.D. Relaxation methods. In Technique of Organic Chemistry (ed., Weissberger, A.) 895–1054 (Interscience, New York, 1963).

    Google Scholar 

  4. Turner, D.H., Flynn, G.W., Lundberg, S.K., Faller, L.D. & Sutin, N. Dimerization of proflavin by the laser Raman temperature-jump method. Nature 239, 215–217 (1972).

    Article  CAS  Google Scholar 

  5. Jennings, P.A. & Wright, P. Formation of a molten globule intermediate early in the kinetic folding pathway of apomyoglobin. Science 262, 892–895 (1993).

    Article  CAS  Google Scholar 

  6. Phillips, C.M., Mizutani, Y. & Hochstrasser, R.M. Ultrafast thermally induced unfolding of RNase A. Proc. Natl. Acad. Sci. USA 92, 7292–7296 (1995).

    Article  CAS  Google Scholar 

  7. Jacob, M. et al. Microsecond folding of the cold shock protein measured by a pressure-jump technique. Biochemistry 38, 2882–2891 (1999).

    Article  CAS  Google Scholar 

  8. Mitra, L. et al. V-i-value analysis: a pressure-based method for mapping the folding transition state ensemble of proteins. J. Am. Chem. Soc. 129, 14108–14109 (2007).

    Article  CAS  Google Scholar 

  9. Ballew, R.M., Sabelko, J., Reiner, C. & Gruebele, M. A single-sweep, nanosecond time resolution laser temperature-jump apparatus. Rev. Sci. Instrum. 67, 3694–3699 (1996).

    Article  CAS  Google Scholar 

  10. Royer, C.A. The nature of the transition state ensemble and the mechanisms of protein folding: A review. Arch. Biochem. Biophys. 469, 34–45 (2008).

    Article  CAS  Google Scholar 

  11. Pearson, D.S., Swartz, D.R. & Geeves, M.A. Fast pressure jumps can perturb calcium and magnesium binding to troponin C F29W. Biochemistry 47, 12146–12158 (2008).

    Article  CAS  Google Scholar 

  12. Seddon, J.M. et al. Pressure-jump X-ray studies of liquid crystal transitions in lipids. Philos. Transact. A Math. Phys. Eng. Sci. 364, 2635–2655 (2006).

    Article  CAS  Google Scholar 

  13. Winter, R. & Dzwolak, W. Exploring the temperature-pressure configurational landscape of biomolecules: from lipid membranes to proteins. Philos. Transact. A Math. Phys. Eng. Sci. 363, 537–562 (2005).

    Article  CAS  Google Scholar 

  14. Kegeles, G. & Ke, C.-H. A light-scattering pressure-jump kinetics apparatus. Anal. Biochem. 68, 138–147 (1975).

    Article  CAS  Google Scholar 

  15. Clegg, R.M., Elson, E.L. & Maxfield, B.W. New technique for optical observation of the kinetics of chemical reactions perturbed by small pressure changes. Biopolymers 14, 883–887 (1975).

    Article  CAS  Google Scholar 

  16. Knoche, W. & Wiese, G. Improved apparatus for pressure-jump relaxation measurements. Chemical Instrumentation 5, 91–98 (1973).

    Article  CAS  Google Scholar 

  17. Magg, C., Kubelka, J., Holtermann, G., Haas, E. & Schmid, F.X. Specificity of the initial collapse in the folding of the cold shock protein. J. Mol. Biol. 360, 1067–1080 (2006).

    Article  CAS  Google Scholar 

  18. Duan, Y. & Kollman, P.A. Pathways to a protein folding intermediate observed in a 1-microsecond simulation in aqueous solution. Science 282, 740–744 (1998).

    Article  CAS  Google Scholar 

  19. Snow, C.D., Nguyen, H., Pande, V. & Gruebele, M. Absolute comparison of simulated and experimental protein folding dynamics. Nature 420, 102–106 (2002).

    Article  CAS  Google Scholar 

  20. Paschek, D., Hempel, S. & Garcia, A.E. Computing the stability diagram ofthe Trp-cage miniprotein. Proc. Natl. Acad. Sci. USA 105, 17754–17759 (2008).

    Article  CAS  Google Scholar 

  21. Kubelka, J., Hofrichter, J. & Eaton, W.A. The protein folding 'speed limit'. Curr. Opin. Struct. Biol. 14, 76–88 (2004).

    Article  CAS  Google Scholar 

  22. Yang, W.Y. & Gruebele, M. Folding at the speed limit. Nature 423, 193–197 (2003).

    Article  CAS  Google Scholar 

  23. Freddolino, P.L., Liu, F., Gruebele, M. & Schulten, K. Ten-microsecond molecular dynamics simulation of a fast-folding WW domain. Biophys. J. 94, L75–L77 (2008).

    Article  CAS  Google Scholar 

  24. Snow, C.D., Sorin, E.J., Rhee, Y.M. & Pande, V.S. How well can simulation predict protein folding kinetics and thermodynamics. Annu. Rev. Biophys. Biomol. Struct. 34, 43–69 (2005).

    Article  CAS  Google Scholar 

  25. Pitera, J.W., Swope, W.C. & Abraham, F.F. Observation of noncooperative folding thermodynamics in simulations of 1BBL. Biophys. J. 94, 4837–4846 (2008).

    Article  CAS  Google Scholar 

  26. Yang, W.Y. & Gruebele, M. Rate-temperature relationships in lambda repressor fragment 6–85 folding. Biochemistry 43, 13018–13025 (2004).

    Article  CAS  Google Scholar 

  27. Dumont, C. et al. Solvent-tuning collapse and helix formation time scales of lambda6–85. Protein Sci. 15, 2596–2604 (2006).

    Article  CAS  Google Scholar 

  28. Yang, W.Y. & Gruebele, M. Kinetic equivalence of the heat and cold structural transitions of lambda6–85. Phil. Trans. R. Soc. Lond. B 43, 13018–13025 (2005).

    Google Scholar 

  29. 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).

    Article  CAS  Google Scholar 

  30. Camilloni, C. et al. Urea and guanidinium chloride denature protein L in different ways in molecular dynamics simulations. Biophys. J. 94, 4654–4661 (2008).

    Article  CAS  Google Scholar 

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Acknowledgements

We acknowledge funding from the National Science Foundation (MCB 0613643), assistance with steady-state data collection by S. Ates, help with the prototype setup by J. Ervin and J. Lee and helpful discussions with H. Bohr. T. Oas (Duke University) provided the pseudo-wild-type lambda repressor fragment plasmid as a gift.

Author information

Authors and Affiliations

  1. Department of Physics, University of Illinois, Urbana, Illinois, USA

    Charles Dumont & Martin Gruebele

  2. Department of Chemistry, University of Illinois, Urbana, Illinois, USA

    Tryggvi Emilsson & Martin Gruebele

  3. Center for Biophysics and Computational Biology, University of Illinois, Urbana, Illinois, USA

    Martin Gruebele

Authors
  1. Charles Dumont
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  2. Tryggvi Emilsson
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  3. Martin Gruebele
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Contributions

C.D. performed the experiments and did data analysis. T.E. designed and made experimental components. M.G. designed the experiment, performed data analysis and wrote the article.

Corresponding author

Correspondence to Martin Gruebele.

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Dumont, C., Emilsson, T. & Gruebele, M. Reaching the protein folding speed limit with large, sub-microsecond pressure jumps. Nat Methods 6, 515–519 (2009). https://doi.org/10.1038/nmeth.1336

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