Folding dynamics of the B1 domain of protein C explored by ultrarapid mixing
Folding dynamics of the B1 domain of protein C explored by ultrarapid mixing
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DOI：
10.1038/13311
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发表时间：
1999-10-01
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影响因子：
--
通讯作者：
Roder, H
中科院分区：
文献类型：
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作者：
Park, SH;Shastry, MCR;Roder, H
对于许多蛋白质来说，紧凑的构象会在折叠限速步骤之前积累。为了了解这些早期构象事件的性质和意义，我们采用超快速混合方法来全面表征蛋白 G 的 57 个残基 B1 结构域的折叠动力学。连续荧光测量在亚毫秒时间尺度 (600-700 mu s) 上表现出主要指数阶段，随后是通过传统停流测量可观察到的具有变性剂依赖性时间常数 (2-30 ms) 的较慢阶段。组合的动力学迹线定量地解释了折叠时 Trp 43 荧光的总变化，包括先前未解析的“爆发相”信号。两个速率常数及其相对振幅的变性依赖性与三态机制完全一致，U 可逆箭头 I 可逆箭头 N，其中 I 是具有类似天然荧光特性的高效中间体。初始折叠阶段相对较慢的速率和指数时间进程表明，在链缩合过程中遇到了相当大的自由能势垒，导致与初始未折叠构象不同的部分组织的状态集合。
For many proteins, compact conformations are known to accumulate in advance of the rate-limiting step in folding. To understand the nature and significance of these early conformational events, we employed ultrarapid mixing methods to fully characterize the kinetics of folding of the 57-residue B1 domain of protein G. Continuous-now fluorescence measurements exhibit a major exponential phase on the submillisecond time scale (600-700 mu s), which is followed by a slower phase with a denaturant-dependent time constant (2-30 ms) observable by conventional stopped-flow measurements. The combined kinetic traces quantitatively account for the total change in Trp 43 fluorescence upon folding, including the previously unresolved 'burst phase' signal. The denaturant dependence of the two rate constants and their relative amplitudes are fully consistent with a three-state mechanism,U reversible arrow I reversible arrow N, where I is a productive intermediate with native-like fluorescence properties. The relatively slow rate and exponential time course of the initial folding phase indicates that a substantial free energy barrier is encountered during chain condensation, resulting in a partially organized ensemble of states distinct from the initial unfolded conformations.