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Tracking the structural dynamics of proteins in solution using time-resolved wide-angle X-ray scattering

An Erratum to this article was published on 01 November 2008

This article has been updated

Abstract

We demonstrate tracking of protein structural changes with time-resolved wide-angle X-ray scattering (TR-WAXS) with nanosecond time resolution. We investigated the tertiary and quaternary conformational changes of human hemoglobin under nearly physiological conditions triggered by laser-induced ligand photolysis. We also report data on optically induced tertiary relaxations of myoglobin and refolding of cytochrome c to illustrate the wide applicability of the technique. By providing insights into the structural dynamics of proteins functioning in their natural environment, TR-WAXS complements and extends results obtained with time-resolved optical spectroscopy and X-ray crystallography.

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Figure 1: Plausibility of TR-WAXS based on photon statistics considerations.
Figure 2: TR-WAXS methodology and data processing.
Figure 3: TR-WAXS data on hemoglobin and the solvent heating contribution.
Figure 4: Comparison between TR-WAXS scattering differences and scattering differences calculated from crystallographic structures.
Figure 5: Application of TR-WAXS to track folding of Cyt-c.

Change history

  • 29 September 2008

    NOTE: In the version of this article initially published, the time scale reported in the Figure 2d legend is incorrect. The correct time scale should be 3 μs. Additionally, the time delay of 320 ms reported in Figure 5b is incorrect. The correct time delay is 200 ms. These errors have been corrected in the HTML and PDF versions of the article.

References

  1. 1

    Moffat, K. Ultrafast time-resolved crystallography. Nat. Struct. Biol. 5, 641–643 (1998).

    CAS  Article  Google Scholar 

  2. 2

    Schotte, F. et al. Watching a protein as it functions with 150-ps time-resolved X-ray crystallography. Science 300, 1944–1947 (2003).

    CAS  Article  Google Scholar 

  3. 3

    Ihee, H. et al. Visualizing reaction pathways in photoactive yellow protein from nanoseconds to seconds. Proc. Natl. Acad. Sci. USA 102, 7145–7150 (2005).

    CAS  Article  Google Scholar 

  4. 4

    Grishaev, A., Wu, J., Trewhella, J. & Bax, A. Refinement of multidomain protein structures by combination of solution small-angle X-ray scattering and NMR data. J. Am. Chem. Soc. 127, 16621–16628 (2005).

    CAS  Article  Google Scholar 

  5. 5

    Xu, X. et al. Dynamics in a pure encounter complex of two proteins studied by solution scattering and paramagnetic NMR spectroscopy. J. Am. Chem. Soc. 130, 6395–6403 (2008).

    CAS  Article  Google Scholar 

  6. 6

    Zuo, X. et al. Global molecular structure and interfaces: refining an RNA:RNA complex structure using solution X-ray scattering data. J. Am. Chem. Soc. 130, 3292–3293 (2008).

    CAS  Article  Google Scholar 

  7. 7

    Hirai, M., Iwase, H., Hayakawa, T., Miura, K. & Inoue, K. Structural hierarchy of several proteins observed by wide-angle solution scattering. J. Synchrotron Radiat. 9, 202–205 (2002).

    CAS  Article  Google Scholar 

  8. 8

    Menk, R.H. et al. Novel detector systems for time resolved SAXS experiments. J. Appl. Crystallogr. 33, 778–781 (2000).

    CAS  Article  Google Scholar 

  9. 9

    Akiyama, S. et al. Conformational landscape of cytochrome c folding studied by microsecond-resolved small-angle X-ray scattering. Proc. Natl. Acad. Sci. USA 99, 1329–1334 (2002).

    CAS  Article  Google Scholar 

  10. 10

    Kainosho, M. et al. Optimal isotope labelling for NMR protein structure determinations. Nature 440, 52–57 (2006).

    CAS  Article  Google Scholar 

  11. 11

    Hofrichter, J., Sommer, J.H., Henry, E.R. & Eaton, W.A. Nanosecond absorption spectroscopy of hemoglobin: elementary processes in kinetic cooperativity. Proc. Natl. Acad. Sci. USA 80, 2235–2239 (1983).

    CAS  Article  Google Scholar 

  12. 12

    Balakrishnan, G. et al. Time-resolved absorption and UV resonance Raman spectra reveal stepwise formation of T quaternary contacts in the allosteric pathway of hemoglobin. J. Mol. Biol. 340, 843–856 (2004).

    CAS  Article  Google Scholar 

  13. 13

    Svergun, D.I. et al. Shape determination from solution scattering of biopolymers. J. Appl. Crystallogr. 30, 798–802 (1997).

    Article  Google Scholar 

  14. 14

    Makowski, L. et al. Molecular crowding inhibits intramolecular breathing motions in proteins. J. Mol. Biol. 375, 529–546 (2008).

    CAS  Article  Google Scholar 

  15. 15

    Bellelli, A., Brunori, M., Miele, A.E., Panetta, G. & Vallone, B. The allosteric properties of hemoglobin: insights from natural and site directed mutants. Curr. Protein Pept. Sci. 7, 17–45 (2006).

    CAS  Article  Google Scholar 

  16. 16

    Eaton, W.A. et al. Evolution of allosteric models for hemoglobin. IUBMB Life 59, 586–599 (2007).

    CAS  Article  Google Scholar 

  17. 17

    Svergun, D.I., Barberato, C. & Koch, M.H.J. CRYSOL - a program to evaluate X-ray solution scattering of biological macromolecules from atomic coordinates. J. Appl. Crystallogr. 28, 768–773 (1995).

    CAS  Article  Google Scholar 

  18. 18

    Svergun, D.I. & Koch, M.H.J. Small-angle scattering studies of biological macromolecules in solution. Rep. Prog. Phys. 66, 1735–1782 (2003).

    CAS  Article  Google Scholar 

  19. 19

    Sawicki, C.A. & Gibson, Q.H. Quaternary conformational changes in human hemoglobin studied by laser photolysis of carboxyhemoglobin. J. Biol. Chem. 251, 1533–1542 (1976).

    CAS  PubMed  Google Scholar 

  20. 20

    Cammarata, M. et al. Impulsive solvent heating probed by picosecond X-ray diffraction. J. Chem. Phys. 124, 124504(1)–124504(9) (2006).

    Article  Google Scholar 

  21. 21

    Fader, W.J. Density perturbations caused by weak absorption of a laser pulse. J. Appl. Phys. 47, 1975–1978 (1976).

    Article  Google Scholar 

  22. 22

    Eaton, W.A., Henry, E.R. & Hofrichter, J. Application of linear free energy relations to protein conformational changes: the quaternary structural change of hemoglobin. Proc. Natl. Acad. Sci. USA 88, 4472–4475 (1991).

    CAS  Article  Google Scholar 

  23. 23

    Fermi, G., Perutz, M.F., Shaanan, B. & Fourme, R. The crystal structure of human deoxyhaemoglobin at 1.74 Angstrom resolution. J. Mol. Biol. 175, 159–174 (1984).

    CAS  Article  Google Scholar 

  24. 24

    Silva, M.M., Rogers, P.H. & Arnone, A. A third quaternary structure of human hemoglobin A at 1.7 Angstrom resolution. J. Biol. Chem. 267, 17248–17256 (1992).

    CAS  PubMed  Google Scholar 

  25. 25

    Lukin, J.A. et al. Quaternary structure of hemoglobin in solution. Proc. Natl. Acad. Sci. USA 100, 517–520 (2003).

    CAS  Article  Google Scholar 

  26. 26

    Srinivasan, R. & Rose, G.D. The T-to-R transformation in hemoglobin: a reevaluation. Proc. Natl. Acad. Sci. USA 91, 11113–11117 (1994).

    CAS  Article  Google Scholar 

  27. 27

    Safo, M.K. & Abraham, D.J. The enigma of the liganded hemoglobin end state: a novel quaternary structure of human carbonmonoxy hemoglobin. Biochemistry 44, 8347–8359 (2005).

    CAS  Article  Google Scholar 

  28. 28

    Guallar, V., Jarzecki, A.A., Friesner, R.A. & Spiro, T.G. Modeling of ligation-induced helix/loop displacements in myoglobin: toward an understanding of hemoglobin allostery. J. Am. Chem. Soc. 128, 5427–5435 (2006).

    CAS  Article  Google Scholar 

  29. 29

    Kachalova, G.S., Popov, A.N. & Bartunik, H.D. A steric mechanism for inhibition of CO binding to heme proteins. Science 284, 473–476 (1999).

    CAS  Article  Google Scholar 

  30. 30

    Jones, C.M. et al. Fast events in protein-folding initiated by nanosecond laser photolysis. Proc. Natl. Acad. Sci. USA 90, 11860–11864 (1993).

    CAS  Article  Google Scholar 

  31. 31

    Arcovito, A., Gianni, S., Brunori, M., Travaglini-Allocatelli, C. & Bellelli, A. Fast coordination changes in cytochrome c do not necessarily imply folding. J. Biol. Chem. 276, 41073–41078 (2001).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank W.A. Eaton and E. Henry for helpful comments, H.-S. Cho and S. Ahn for helpful discussions about the data analysis, and Y.O. Jung, K.H. Kim and J.H. Lee for their assistance with sample preparation and experiments. This research was supported in part by the Intramural Research Program of the National Institutes of Health to P.A.A., by EU grant FLASH: FP6-503641 to M.W., and a grant from the Creative Research Initiatives (Center for Time-Resolved Diffraction) of the Ministry of Education, Science and Technology, Korea Science and Engineering Foundation to H.I.

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Correspondence to Marco Cammarata or Hyotcherl Ihee.

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Supplementary Figures 1–6, Supplementary Tables 1–4, Supplementary Methods (PDF 975 kb)

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Cammarata, M., Levantino, M., Schotte, F. et al. Tracking the structural dynamics of proteins in solution using time-resolved wide-angle X-ray scattering. Nat Methods 5, 881–886 (2008). https://doi.org/10.1038/nmeth.1255

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