Kinetic and Structural Analysis of Submillisecond Folding Events in Cytochrome c
Kinetic and Structural Analysis of Submillisecond Folding Events in Cytochrome c
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细胞色素 c 中亚毫秒折叠事件的动力学和结构分析
DOI：
10.1002/chin.199905294
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
H. Roder
中科院分区：
文献类型：
--
作者：
M. Shastry;J. M. Sauder;H. Roder
近年来，人们对肽和蛋白质基本构象变化的时间尺度和性质有了很多了解。 1, 2 虽然小蛋白质通常需要几毫秒或更长的时间才能完成折叠过程，但表明局部和远程三级结构重排的光谱变化通常被发现发生在更短的时间范围内。然而，这些观察的意义仍然是一个激烈争论的话题。 1, 3-9 折叠中第一次大规模构象事件的时间尺度是由什么决定的？多肽链的缩合是由非特异性疏水相互作用还是由更特异性的三级相互作用驱动的？致密态的形成是由链式扩散控制的渐进过程还是受自由能离散势垒限制的二态转变？研究蛋白质折叠动力学最常用的方法依赖于快速混合方法，通过溶剂条件的突然变化启动重折叠，并结合光学检测（吸光度、荧光、圆二色性等）或氢交换标记与 NMR 相结合。尽管传统停流和骤冷仪器的时间分辨率仅限于几毫秒，但人们经常发现观察到的动力学并不能解释与平衡展开转变相关的信号的总变化，这表明在测量的死区时间内发生了一些构象变化。这种所谓的突发相效应已在多种蛋白质中观察到，使用各种光谱参数，包括圆二色性 (CD)、内在和外在荧光探针，以及酰胺质子免受溶剂交换的保护（参考文献 8 和 10-12 中综述）。这些观察结果通常归因于具有熔球某些特征的折叠中间体的快速积累，即具有高水平二级结构的致密态的动态系综。另一方面，也有人提出，爆发相可能只是反映了多肽链对溶剂条件变化的初始响应，这有利于由于非特异性疏水相互作用而形成致密变性状态。 3,6,7图1以简单的动力学方案和各种条件下预期的相应自由能图说明了两种可能的折叠场景。图 A 描绘了一个最小的三态机制，其中折叠从初始的未折叠状态 U，经过必要的中间体 I，到天然状态 N（或后期折叠中间体）。 U 表示溶剂条件变化后立即获得的展开构象的集合（可能包含非随机结构的簇），而 I 表示具有不同光谱特征的部分折叠状态的集合。 I 的形成被描述为快速（亚毫秒）和可逆的预平衡，U h I，它先于 I 到 N 的限速转化。在稳定条件下，I 累积为瞬态动力学中间体，产生双相动力学，具有快速（通常动力学上未解决）瞬态，然后是在停流时间尺度（> 1 ms）上可观察到的较慢相。添加变性剂会降低部分和完全展开态相对于 N 的自由能，具体取决于溶剂可及表面积的相对量。因此，当我们接近展开的过渡区域时，I 的数量会减少，并且慢相以快相为代价获得振幅，这与观察到的突发相一致……
Much has been learned in recent years about the time scale and nature of elementary conformational changes in peptides and proteins. 1, 2 While small proteins typically require milliseconds or longer to complete the process of folding, spectroscopic changes indicative of both local and long-range tertiary structural rearrangements are often found to occur on a much shorter time scale. However, the significance of these observations remains a topic of lively debate. 1, 3-9 What determines the time scale of the first large-scale conformational events in folding? Is the condensation of the polypeptide chain driven by nonspecific hydrophobic interactions or by more specific tertiary interactions? Is the formation of compact states a gradual process governed by chain diffusion or a two-state transition limited by a discrete barrier in free energy? The most commonly used approach for investigating the kinetics of protein folding relies on rapid mixing methods to initiate refolding via a sudden change in solvent conditions, coupled with optical detection (absorbance, fluorescence, circular dichroism, etc.) or hydrogen exchange labeling in conjunction with NMR. Although the time resolution of conventional stopped-flow and quenched-flow instruments is limited to a few milliseconds, one often finds that the observed kinetics does not account for the total change in the signal associated with the equilibrium unfolding transition, indicating that some conformational changes occur within the dead time of the measurement. This so-called burst-phase effect has been observed for numerous proteins, using various spectroscopic parameters, including circular dichroism (CD), intrinsic and extrinsic fluorescence probes, and protection of amide protons against solvent exchange (reviewed in refs 8 and 10-12). These observations are often attributed to the rapid accumulation of folding intermediates with some of the characteristics of a molten globule, ie, a dynamic ensemble of compact states with high levels of secondary structure. On the other hand, it has also been suggested that the burst phase may simply reflect the initial response of the polypeptide chain to the change in solvent conditions, which favors formation of compact denatured states due to nonspecific hydrophobic interactions. 3, 6, 7Two possible folding scenarios are illustrated in Figure 1 in terms of simple kinetic schemes and the corresponding free energy diagrams expected under various conditions. Panel A depicts a minimal three-state mechanism where folding proceeds from the initial unfolded state, U, through an obligatory intermediate, I, to the native state, N (or a late folding intermediate). U represents the ensemble of unfolded conformations obtained immediately after the change in solvent conditions (which may contain clusters of nonrandom structure), whereas I represents an ensemble of partially folded states with distinct spectroscopic characteristics. The formation of I is described as a rapid (submillisecond) and reversible preequilibrium, U h I, which precedes the rate-limiting conversion of I into N. Under stabilizing conditions, I accumulates as a transient kinetic intermediate, giving rise to biphasic kinetics with a fast (often kinetically unresolved) transient followed by a slower phase observable on the stopped-flow time scale (> 1 ms). Addition of denaturant lowers the free energies of partially and fully unfolded states relative to N, depending on the relative amounts of solvent-accessible surface area. Thus, the population of I decreases as we approach the unfolding transition region, and the slow phase gains amplitude at the expense of the fast phase, which is consistent with the observed burst-phase …
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荚膜红细菌细胞色素c2折叠和展开的动力学机制。
DOI：
10.1021/bi961976k
发表时间：
1996
期刊：
Biochemistry.
影响因子：
--
作者：
Sauder,JM;MacKenzie,NE;Roder,H
通讯作者：
Roder,H
快速折叠实验和蛋白质折叠能量景观的地形。
影响因子：
--
作者：
Wolynes,P;Luthey-Schulten,Z;Onuchic,J
通讯作者：
Onuchic,J
影响因子：
2.9
作者：
W. F. Walkenhorst;S. Green;H. Roder
通讯作者：
W. F. Walkenhorst;S. Green;H. Roder
细胞色素 c 早期折叠中间体中的酰胺保护。
DOI：
10.1016/s1359-0278(98)00040-6
发表时间：
1998
期刊：
Folding & design.
影响因子：
--
作者：
Sauder,JM;Roder,H
通讯作者：
Roder,H
影响因子：
2.9
作者：
Colon, W;Wakem, LP;Roder, H
通讯作者：
Roder, H