文章基本信息

标题：Snapshots of a protein folding intermediate
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作者：Seiji Yamada ; Nicole D. Bouley Ford ; Gretchen E. Keller 等
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：2013
卷号：110
期号：5
页码：1606-1610
DOI：10.1073/pnas.1221832110
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：We have investigated the folding dynamics of Thermus thermophilus cytochrome c552 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 {beta}-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-s 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.
关键词：FRET ; microfluidic mixing ; protein refolding