Stability and Folding Kinetics of a Ubiquitin Mutant with a Strong Propensity for Nonnative -Hairpin Conformation in

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• 作者：Geoffrey W. Platt ; Stephen A. Simpson ; Robert Layfield ; and Mark S. Searle
• 刊名：Biochemistry
• 出版年：2003
• 出版时间：November 25, 2003
• 年：2003
• 卷：42
• 期：46
• 页码：13762 - 13771
• 全文大小：182K
• 年卷期：v.42,no.46(November 25, 2003)
• ISSN：1520-4995

文摘

A F45W mutant of yeast ubiquitin has been used as a model system to examine the effects ofnonnative local interactions on protein folding and stability. Mutating the native TLTGK G-bulged typeI turn in the N-terminal mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-hairpin to NPDG stabilizes a nonnative mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-strand alignment in the isolatedpeptide fragment. However, NMR structural analysis of the native and mutant proteins shows that theNPDG mutant is forced to adopt the native mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-strand alignment and an unfavorable type I NPDG turn.The mutant is significantly less stable (~9 kJ mol^-1) and folds 30 times slower than the native sequence,demonstrating that local interactions can modulate protein stability and that attainment of a nativelikemages/gifchars/beta2.gif" BORDER=0 ALIGN="middle">-hairpin conformation in the transition state ensemble is frustrated by the turn mutations. Surprising,alcoholic cosolvents [5-10% (v/v) TFE] are shown to accelerate the folding rate of the NPDG mutant.We conclude, backed-up by NMR data on the peptide fragments, that even though nonnative states in thedenatured ensemble are highly populated and their stability further enhanced in the presence of cosolvents,the simultaneous increase in the proportion of nativelike secondary structure (hairpin or helix), in rapidequilibrium with nonnative states, is sufficient to accelerate the folding process. It is evident that modulatinglocal interactions and increasing nonnative secondary structure propensities can change protein stabilityand folding kinetics. However, nonlocal contacts formed in the global cooperative folding event appearto determine structural specificity.