Probing the protein-folding mechanism using denaturant and temperature effects on rate constants

Guinn E.J.; Kontur W.S.; Tsodikov O.V.Shkel I.Record Jr. M.T.

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Probing the protein-folding mechanism using denaturant and temperature effects on rate constants

机译：Probing the protein-folding mechanism using denaturant and temperature effects on rate constants

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Protein folding has been extensively studied, but many questions remain regarding the mechanism. Characterizing early unstable intermediates and the high-free-energy transition state (TS) will help answer some of these. Here, we use effects of denaturants (urea, guanidinium chloride) and temperature on folding and unfolding rate constants and the overall equilibrium constant as probes of surface area changes in protein folding. We interpret denaturant kinetic m-values and activation heat capacity changes for 13 proteins to determine amounts of hydrocarbon and amide surface buried in folding to and from TS, and for complete folding. Predicted accessible surface area changes for complete folding agree in most cases with structurally determined values. We find that TS is advanced (50-90 of overall surface burial) and that the surface buried is disproportionately amide, demonstrating extensive formation of secondary structure in early intermediates. Models of possible pre-TS intermediates with all elements of the native secondary structure, created for several of these proteins, bury less amide and hydrocarbon surface than predicted for TS. Therefore, we propose that TS generally has both the native secondary structure and sufficient organization of other regions of the backbone to nucleate subsequent (post-TS) formation of tertiary interactions. The approach developed here provides proof of concept for the use of denaturants and other solutes as probes of amount and composition of the surface buried in coupled folding and other large conformational changes in TS and intermediates in protein processes.

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《Proceedings of the National Academy of Sciences of the United States of America》 |2013年第42期|16784-16789|共6页
作者
Guinn E.J.; Kontur W.S.; Tsodikov O.V.Shkel I.Record Jr. M.T.;
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作者单位

Department of Chemistry, University of Wisconsin, Madison, WI 53706, United States;

Department of Bacteriology, University of Wisconsin, Madison, WI 53706, United States;

Department of Pharmaceutical Sciences, University of Kentucky, Lexington, KY 40536, United States;

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收录信息美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
原文格式 PDF
正文语种英语
中图分类自然科学总论;
关键词
Collapse; Kinetics; Native fold; Protein denaturation; Stability;

Probing the protein-folding mechanism using denaturant and temperature effects on rate constants

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