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Published January 29, 2013 | Published + Supplemental Material
Journal Article Open

Snapshots of a protein folding intermediate


We have investigated the folding dynamics of Thermus thermophilus cytochrome c_(552) by time-resolved fluorescence energy transfer between the heme and each of seven site-specific fluorescent probes. We have found both an equilibrium unfolding intermediate and a distinct refolding intermediate from kinetics studies. Depending on the protein region monitored, we observed either two-state or three-state denaturation transitions. The unfolding intermediate associated with three-state folding exhibited native contacts in β-sheet and C-terminal helix regions. We probed the formation of a refolding intermediate by time-resolved fluorescence energy transfer between residue 110 and the heme using a continuous flow mixer. The intermediate ensemble, a heterogeneous mixture of compact and extended polypeptides, forms in a millisecond, substantially slower than the ∼100-μs formation of a burst-phase intermediate in cytochrome c. The surprising finding is that, unlike for cytochrome c, there is an observable folding intermediate, but no microsecond burst phase in the folding kinetics of the structurally related thermostable protein.

Additional Information

© 2013 National Academy of Sciences. Contributed by Harry B. Gray, December 13, 2012 (sent for review May 29, 2012). Published online before print January 14, 2013. S.Y. thanks Dr. Julie A. Hoy (Caltech) for X-ray data collection; Dr. Hiroshi Nakajima (Nagoya University) for advice on expression and purification of c552; and Drs. Yuichi Tokita and Yoshio Goto (Sony Corporation) for the management, promotion, and kind support of this study. S.Y. was supported by a Research Fellowship of Sony Corporation. Research at Caltech was supported by National Institutes of Health Grants DK019038 (to H.B.G. and J.R.W.) and GM068461 (to J.R.W.). Author contributions: S.Y., N.D.B.F., H.B.G., and J.R.W. designed research; S.Y. and N.D.B.F. performed research; S.Y., N.D.B.F., W.C.F., H.B.G., and J.R.W. analyzed data; and S.Y., N.D.B.F., G.E.K., H.B.G., and J.R.W. wrote the paper.

Attached Files

Published - PNAS-2013-Yamada-1606-10.pdf

Supplemental Material - pnas.201221832SI.pdf


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