Evidence for barrier-limited protein folding kinetics on the microsecond time scale

Evidence for barrier-limited protein folding kinetics on the microsecond time scale
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微秒时间尺度上屏障限制的蛋白质折叠动力学的证据

DOI:
10.1038/nsb0598-385
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发表时间:
1998
期刊:
Nature Structural Biology
影响因子:
--
通讯作者:
H. Roder
H. Roder
中科院分区:
--
文献类型:
--
作者:
M. Shastry;H. Roder

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虽然蛋白质折叠中的重要结构事件发生在亚毫秒级的时间尺度上,但传统动力学方法有限的时间分辨率排除了对多肽链初始崩溃的直接观察。我们最近发展的一种连续流动毛细管混合方法使我们能够解释∼5-10-5-102Trp 5-10-5-102Trp 5-10-5-102Trp 5-10-5-102Trp 5-10-5-102Trp 5-10-5-102Trp 5-10-5-102Trp 5-10-5-102Trp 5-10-5-10 2的全部荧光变化,包括先前未分辨的Trp 59荧光猝灭(爆发相),表明致密状态的形成。折叠动力学表现出一个主要的指数过程,其时间常数为∼50μS,与初始条件和血红素连接状态无关,表明在多肽链的初始折叠过程中遇到了共同的自由能垒。由此产生的松散堆积的中间体在特定三级相互作用的限速形成之前积累,证实了先前的迹象,即折叠至少涉及两个不同的阶段。
Although important structural events in protein folding are known to occur on the submillisecond time scale, the limited time resolution of conventional kinetic methods has precluded direct observation of the initial collapse of the polypeptide chain. A continuous-flow capillary mixing method recently developed by us made it possible to account for the entire fluorescence change associated with refolding of cytochrome c from ∼5–10-5-102 s, including the previously unresolved quenching of Trp 59 fluorescence (burst phase) indicative of the formation of compact states. The kinetics of folding exhibits a major exponential process with a time constant of ∼50 μs, independent of initial conditions and heme ligation state, indicating that a common free energy barrier is encountered during the initial collapse of the polypeptide chain. The resulting loosely packed intermediate accumulates prior to the rate-limiting formation of specific tertiary interactions, confirming previous indications that folding involves at least two distinct stages.
DOI: 10.1073/pnas.94.5.1779
发表时间: 1997-03-04
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