Earliest events in protein folding:: Submicrosecond secondary structure formation in reduced cytochrome c
Earliest events in protein folding:: Submicrosecond secondary structure formation in reduced cytochrome c
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DOI：
10.1021/jp030006l
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发表时间：
2003-10-09
影响因子：
2.9
通讯作者：
Kliger, DS
中科院分区：
文献类型：
--
作者：
Chen, EF;Goldbeck, RA;Kliger, DS
蛋白质折叠是一个动态的过程，其独特的特征是未折叠状态的巨大构象异质性。这种构象异质性意味着在折叠动力学中可能存在显著的动力学异质性，然而许多折叠反应似乎是均匀进行的，即，从彼此平衡的未折叠构象和过渡状态。在到达折叠状态的过程中，在什么时间点，未折叠状态的动力学异质性丧失为构象平衡一直是一个悬而未决的问题。在这方面，我们提出的证据，从纳秒远紫外旋光色散光谱的最快的折叠过程中观察到的光还原细胞色素c，二级结构形成过程中进行的亚微秒时间尺度在高变性剂浓度（观察到的“突发”相毫秒停流圆二色性研究）。这种快速折叠过程的动力学意味着，在完全还原样品所需的时间（约100 μ s）内，它是从与大量蛋白质构象异构体不平衡的构象系综进行的。因此，我们确定了构象平衡所需时间的下限约为10（-4）s，与先前从光解细胞色素c-CO的时间分辨磁性圆二色性测量的估计一致（戈德贝克，R.一、托马斯，Y. G.地; Chen，E.;埃斯奎拉河M.的; Kliger，D. S. Proc. Natl. Acad. Sci. 1999，96，2782-2787）。结合停流测量的上限（Lyubovitsky，J.G.;格雷，H. B.;温克勒，J.R. J. Am. Soc.2002，124，5481-5495），这允许我们将构象扩散时间括起来，即，通过构象平衡，该蛋白质未折叠状态的动力学不均一性消失的时间间隔，在类似于10（-4）-10（-3）s的范围内。构象扩散时间的这种估计意味着最早的折叠事件，螺旋形成和可能的扩展构象的崩溃，在能量景观制度下进行，其中构象扩散是缓慢的，而最终（毫秒）折叠阶段进行沿着经典的动力学途径。
Protein folding is uniquely characterized as a dynamic process by the tremendous conformational heterogeneity of the unfolded state. This conformational heterogeneity implies the possibility of significant kinetic heterogeneity in the folding dynamics, yet many folding reactions appear to proceed homogeneously, i.e., from unfolded conformations that are in equilibrium with each other and a transition state. At what point in time on the way to the folded state the kinetic heterogeneity of the unfolded state is lost to conformational equilibration has been an open question. We present evidence in this regard obtained from nanosecond far-UV optical rotatory dispersion spectroscopy of the fastest folding process observed in photoreduced cytochrome c, a secondary structure formation process proceeding on a submicrosecond time scale at high denaturant concentration (observed as a "burst" phase in millisecond stopped-flow circular dichroism studies). The kinetics of this fast folding process imply that it proceeds from a conformational ensemble that is not in equilibrium with the bulk of protein conformers during the time required to completely reduce the sample, similar to100 mus. We thus determine a lower limit on the time required for conformational equilibration of similar to10(-4) s, in agreement with the previous estimate from time-resolved magnetic circular dichroism measurements on photolyzed cytochrome c-CO (Goldbeck, R. A.; Thomas, Y. G.; Chen, E.; Esquerra, R. M.; Kliger, D. S. Proc. Natl. Acad. Sci. 1999, 96, 2782-2787). Combined with an upper limit from stopped-flow measurements (Lyubovitsky, J. G.; Gray, H. B.; Winkler, J. R. J. Am. Chem. Soc. 2002, 124, 5481-5495), this allows us to bracket the conformational diffusion time, i.e., the time interval over which the kinetic heterogeneity of this protein's unfolded state is lost through conformational equilibration, within the range similar to10(-4)-10(-3) s. This estimate of the conformational diffusion time implies that the earliest folding events, helix formation and possibly the collapse of extended conformations, proceed under an energy landscape regime wherein conformational diffusion is slow, whereas the final (milliseconds) folding phase proceeds along a classical kinetic pathway.