Abstract
Time resolution of current single-molecule fluorescence techniques is limited to milliseconds because of dye blinking and bleaching. Here we introduce a photoprotection strategy that affords microsecond resolution by combining efficient triplet quenching by oxygen and Trolox with minimized bleaching via the oxygen radical scavenger cysteamine. Using this approach we resolved the single-molecule microsecond conformational fluctuations of two proteins: the two-state folder α-spectrin SH3 domain and the ultrafast downhill folder BBL.
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References
Moerner, W.E. Proc. Natl. Acad. Sci. USA 104, 12596–12602 (2007).
Roy, R., Hohng, S. & Ha, T. Nat. Methods 5, 507–516 (2008).
Schuler, B. & Eaton, W.A. Curr. Opin. Struct. Biol. 18, 16–26 (2008).
Gell, C., Brockwell, D. & Smith, A. Handbook of Single Molecule Fluorescence Spectroscopy. (Oxford University Press, 2006).
Kong, X.X., Nir, E., Hamadani, K. & Weiss, S. J. Am. Chem. Soc. 129, 4643–4654 (2007).
Rasnik, I., McKinney, S.A. & Ha, T. Nat. Methods 3, 891–893 (2006).
Turro, N.J. Modern Molecular Photochemistry. (University Science Books, 1991).
Dittrich, P.S. & Schwille, P. Appl. Phys. B 73, 829–837 (2001).
Misik, V., Miyoshi, N. & Riesz, P. Free Radic. Biol. Med. 26, 961–967 (1999).
Wozniak, A.K., Schroder, G.F., Grubmuller, H., Seidel, C.A. & Oesterhelt, F. Proc. Natl. Acad. Sci. USA 105, 18337–18342 (2008).
Li, P., Oliva, F.Y., Naganathan, A.N. & Muñoz, V. Proc. Natl. Acad. Sci. USA 106, 103–108 (2009).
Muñoz, V. Annu. Rev. Biophys. Biomol. Struct. 36, 395–412 (2007).
Eaton, W.A. et al. Annu. Rev. Biophys. Biomol. Struc. 29, 327–359 (2000).
Fawni, N.L., Douclef, M., Suh, J.Y. & Clore, M.G. Proc. Natl. Acad. Sci. USA 107, 1379–1384 (2010).
Neuweiler, H., Johnson, C.M. & Fersht, A.R. Proc. Natl. Acad. Sci. USA 106, 18569–18574 (2009).
Morgan, M.A., Okamoto, K., Kahn, J.D. & English, D.S. Biophys. J. 89, 2588–2596 (2005).
Gopich, I.V. & Szabo, A. J. Phys. Chem. B 109, 17683–17688 (2005).
McKinney, S.A., Joo, C. & Ha, T. Biophys. J. 91, 1941–1951 (2006).
Lapidus, L.J., Steinbach, P.J., Eaton, W.A., Szabo, A. & Hofrichter, J. J. Phys. Chem. B 106, 11628–11640 (2002).
Acknowledgements
This research was supported in part by the Marie Curie excellence grant MEXT-CT-2006-042334 and grants BFU2008-03237 and CONSOLIDER CSD2009-00088 from the Spanish Ministry of Science and Innovation.
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L.A.C. and J.L. prepared samples, identified the oxygen radical scavengers, performed experiments and analyzed data. L.A.C. performed all the additional experiments requested by the reviewers. X.W. acquired and analyzed data. R.R. performed the stochastic simulations of downhill folding. D.S.E. supervised data acquisition and designed research. V.M. designed research, supervised data acquisition, performed and supervised data analysis and simulations, and wrote the manuscript.
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Campos, L., Liu, J., Wang, X. et al. A photoprotection strategy for microsecond-resolution single-molecule fluorescence spectroscopy. Nat Methods 8, 143–146 (2011). https://doi.org/10.1038/nmeth.1553
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DOI: https://doi.org/10.1038/nmeth.1553
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