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Folding energy landscape of cytochrome cb_(562)

Kimura, Tetsunari and Lee, Jennifer C. and Gray, Harry B. and Winkler, Jay R. (2009) Folding energy landscape of cytochrome cb_(562). Proceedings of the National Academy of Sciences of the United States of America, 106 (19). pp. 7834-7839. ISSN 0027-8424. PMCID PMC2683080. doi:10.1073/pnas.0902562106.

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Cytochrome cb_(562) 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 cb_(562) 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 b_(562) 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.

Item Type:Article
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URLURL TypeDescription CentralArticle
Gray, Harry B.0000-0002-7937-7876
Winkler, Jay R.0000-0002-4453-9716
Additional Information:© 2009 by the National Academy of Sciences. Contributed by Harry B. Gray, March 9, 2009 (received for review February 11, 2009). Published online before print April 28, 2009, doi: 10.1073/pnas.0902562106. 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. We thank Professor Linda Thöny-Meyer (Eidgenössische Technische Hochschule Zürich, Zürich, Switzerland) for the ccm plasmid pEC86. We also thank Ekaterina V. Pletneva for many helpful discussions. This work was supported by National Institutes of Health Grants GM068461 (to J.R.W.) and DK019038 (to H.B.G.) and by an Arnold and Mabel Beckman Foundation Senior Research Fellowship (to J.C.L.). T.K. was supported by Japan Society for the Promotion of Science Postdoctoral Fellowships for Young Scientists and for Research Abroad. The authors declare no conflict of interest.
Funding AgencyGrant Number
Arnold and Mabel Beckman FoundationUNSPECIFIED
Japan Society for the Promotion of Science (JSPS)UNSPECIFIED
Subject Keywords:four-helix bundle; minimal frustration; protein folding; time-resolved fluorescence energy transfer; tryptophan
Issue or Number:19
PubMed Central ID:PMC2683080
Record Number:CaltechAUTHORS:20090813-110702430
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:15009
Deposited By: Tony Diaz
Deposited On:13 Aug 2009 20:52
Last Modified:08 Nov 2021 23:15

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