Folding energy landscape of cytochrome cb_(562)
Abstract
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.
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.Attached Files
Published - Kimura2009p4249P_Natl_Acad_Sci_Usa.pdf
Supplemental Material - Kimura2009p4249P_Natl_Acad_Sci_Usa_appendix.pdf
Supplemental Material - Kimura2009p4249P_Natl_Acad_Sci_Usa_supp.pdf
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Additional details
- PMCID
- PMC2683080
- Eprint ID
- 15009
- Resolver ID
- CaltechAUTHORS:20090813-110702430
- GM068461
- NIH
- DK019038
- NIH
- Arnold and Mabel Beckman Foundation
- Japan Society for the Promotion of Science (JSPS)
- Created
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2009-08-13Created from EPrint's datestamp field
- Updated
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2021-11-08Created from EPrint's last_modified field