期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2009
卷号：106
期号：19
页码：7834-7839
DOI：10.1073/pnas.0902562106
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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 {micro}s 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.
关键词：four-helix bundle ; minimal frustration ; protein folding ; time-resolved fluorescence energy transfer ; tryptophan
