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• 作者：Osman Bilsel ; Jill A. Zitzewitz ; Katherine E. Bowers ; and C. Robert Matthews
• 刊名：Biochemistry
• 出版年：1999
• 出版时间：January 19, 1999
• 年：1999
• 卷：38
• 期：3
• 页码：1018 - 1029
• 全文大小：146K
• 年卷期：v.38,no.3(January 19, 1999)
• ISSN：1520-4995

文摘

A variety of techniques have been used to investigate the urea-induced kinetic foldingmechanism of the mages/gifchars/alpha.gif" BORDER=0>-subunit of tryptophan synthase from Escherichia coli. A distinctive property of this29 kDa mages/gifchars/alpha.gif" BORDER=0>/mages/gifchars/beta2.gif" BORDER=0 ALIGN="middle"> barrel protein is the presence of two stable equilibrium intermediates, populated at approximately3 and 5 M urea. The refolding process displays multiple kinetic phases whose lifetimes span thesubmillisecond to greater than 100 s time scale; unfolding studies yield two relaxation times on the orderof 10-100 s. In an effort to understand the populations and structural properties of both the stable andtransient intermediates, stopped-flow, manual-mixing, and equilibrium circular dichroism data were globallyfit to various kinetic models. Refolding and unfolding experiments from various initial urea concentrationsas well as forward and reverse double-jump experiments were critical for model discrimination. The simplestkinetic model that is consistent with all of the available data involves four slowly interconverting unfoldedforms that collapse within 5 ms to a marginally stable intermediate with significant secondary structure.This early intermediate is an off-pathway species that must unfold to populate a set of four on-pathwayintermediates that correspond to the 3 M urea equilibrium intermediate. Reequilibrations among theseconformers act as rate-limiting steps in folding for a majority of the population. A fraction of the nativeconformation appears in less than 1 s at 25 mages/entities/deg.gif">C, demonstrating that even large proteins can rapidly traversea complex energy surface.