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Research Article

Folding energy landscape of cytochrome cb562

Tetsunari Kimura, Jennifer C. Lee, Harry B. Gray, and Jay R. Winkler
  1. Beckman Institute, California Institute of Technology, Pasadena, CA 91125-7400

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PNAS May 12, 2009 106 (19) 7834-7839; https://doi.org/10.1073/pnas.0902562106
Tetsunari Kimura
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Jennifer C. Lee
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Harry B. Gray
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  • For correspondence: hbgray@caltech.edu winklerj@caltech.edu
Jay R. Winkler
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  • For correspondence: hbgray@caltech.edu winklerj@caltech.edu
  1. Contributed by Harry B. Gray, March 9, 2009 (received for review February 11, 2009)

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Abstract

Cytochrome cb562 is a variant of an Escherichia coli four-helix bundle b-type heme protein in which the porphyrin prosthetic group is covalently ligated to the polypeptide near the terminus of helix 4. Studies from other laboratories have shown that the apoprotein folds rapidly without the formation of intermediates, whereas the holoprotein loses heme before native structure can be attained. Time-resolved fluorescence energy transfer (TRFET) measurements of cytochrome cb562 refolding triggered using an ultrafast continuous-flow mixer (150 μs dead time) reveal that heme attachment to the polypeptide does not interfere with rapid formation of the native structure. Analyses of the TRFET data produce distributions of Trp-59–heme distances in the protein before, during, and after refolding. Characterization of the moments and time evolution of these distributions provides compelling evidence for a refolding mechanism that does not involve significant populations of intermediates. These observations suggest that the cytochrome b562 folding energy landscape is minimally frustrated and able to tolerate the introduction of substantial perturbations (i.e., the heme prosthetic group) without the formation of deep misfolded traps.

  • four-helix bundle
  • minimal frustration
  • protein folding
  • time-resolved fluorescence energy transfer
  • tryptophan

Footnotes

  • 2To whom correspondence may be addressed. E-mail: hbgray{at}caltech.edu or winklerj{at}caltech.edu
  • Author contributions: T.K., J.C.L., H.B.G., and J.R.W. designed research; T.K. and J.C.L. performed research; T.K., J.C.L., H.B.G., and J.R.W. analyzed data; and T.K., J.C.L., H.B.G., and J.R.W. wrote the paper.

  • ↵1Present address: Laboratory of Molecular Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.

  • The authors declare no conflict of interest.

  • This article contains supporting information online at www.pnas.org/cgi/content/full/0902562106/DCSupplemental.

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Folding energy landscape of cytochrome cb562
Tetsunari Kimura, Jennifer C. Lee, Harry B. Gray, Jay R. Winkler
Proceedings of the National Academy of Sciences May 2009, 106 (19) 7834-7839; DOI: 10.1073/pnas.0902562106

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Folding energy landscape of cytochrome cb562
Tetsunari Kimura, Jennifer C. Lee, Harry B. Gray, Jay R. Winkler
Proceedings of the National Academy of Sciences May 2009, 106 (19) 7834-7839; DOI: 10.1073/pnas.0902562106
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  • Biophysics and Computational Biology
Proceedings of the National Academy of Sciences: 106 (19)
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