Kinetic evidence for folding and unfolding intermediates in staphylococcal nuclease.
Kinetic evidence for folding and unfolding intermediates in staphylococcal nuclease.
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DOI：
10.1021/bi9700476
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发表时间：
1997-05
期刊：
影响因子：
2.9
通讯作者：
W. F. Walkenhorst;S. Green;H. Roder
中科院分区：
文献类型：
--
作者：
W. F. Walkenhorst;S. Green;H. Roder
在蛋白质折叠研究中通常观察到的复杂动力学行为表明，在溶液中存在不均匀的分子群体，并且在未折叠分子向完全天然形式的转化中涉及许多离散步骤。蛋白质折叠中的一个中心问题是这些动力学事件中的任何一个是否代表对有效折叠重要的构象步骤，而不是由缓慢步骤如脯氨酸异构化或多肽链的错误折叠引起的副反应。为了解决这个问题，我们使用停流荧光技术来表征SNase的Pro-变体的折叠和解折叠的动力学机制，其中所有六个脯氨酸残基被甘氨酸或丙氨酸取代。与野生型蛋白相比，其表现出一系列脯氨酸依赖性的缓慢折叠相，Pro-SNase的折叠动力学要简单得多，这使得定量动力学分析成为可能。尽管没有脯氨酸或其他复杂的因素，折叠动力学仍然包含几个阶段，并表现出复杂的变性剂依赖性。GuHCl依赖的主要可观察到的折叠阶段和一个明显的滞后在本机状态的外观提供了明确的证据早期折叠中间。Trp 140在α-螺旋结构域中的荧光对这种早期中间体的形成不敏感，这与具有稳定的β-结构域和很大程度上无序的α-螺旋区域的部分折叠状态一致。需要第二个中间体来模拟Pro变体的解折叠动力学，这显示了在限速解折叠步骤中变性剂诱导的变化的证据。包括这两个中间体，我们能够完全模拟的主要阶段（S）在折叠和展开在广泛的变性剂浓度使用顺序四态折叠机制。为了对前突变体观察到的次要缓慢阶段进行建模，需要含有源自不同未折叠状态的平行途径的六态方案。这种替代未折叠构象的性质与由于存在非脯氨酰顺式肽键而预期的性质一致。为了测试动力学模型，我们使用基于六态方案的模拟，并且能够在变性剂浓度范围内完全再现Pro- SNase的折叠动力学。
The complex kinetic behavior commonly observed in protein folding studies suggests that a heterogeneous population of molecules exists in solution and that a number of discrete steps are involved in the conversion of unfolded molecules to the fully native form. A central issue in protein folding is whether any of these kinetic events represent conformational steps important for efficient folding rather than side reactions caused by slow steps such as proline isomerization or misfolding of the polypeptide chain. In order to address this question, we used stopped-flow fluorescence techniques to characterize the kinetic mechanism of folding and unfolding for a Pro- variant of SNase in which all six proline residues were replaced by glycines or alanines. Compared to the wild-type protein, which exhibits a series of proline-dependent slow folding phases, the folding kinetics of Pro- SNase were much simpler, which made quantitative kinetic analysis possible. Despite the absence of prolines or other complicating factors, the folding kinetics still contain several phases and exhibit a complex denaturant dependence. The GuHCl dependence of the major observable folding phase and a distinct lag in the appearance of the native state provide clear evidence for an early folding intermediate. The fluorescence of Trp140 in the alpha-helical domain is insensitive to the formation of this early intermediate, which is consistent with a partially folded state with a stable beta-domain and a largely disordered alpha-helical region. A second intermediate is required to model the kinetics of unfolding for the Pro- variant, which shows evidence for a denaturant-induced change in the rate-limiting unfolding step. With the inclusion of these two intermediates, we are able to completely model the major phase(s) in both folding and unfolding across a wide range of denaturant concentrations using a sequential four-state folding mechanism. In order to model the minor slow phase observed for the Pro- mutant, a six-state scheme containing a parallel pathway originating from a distinct unfolded state was required. The properties of this alternate unfolded conformation are consistent with those expected due to the presence of a non-prolyl cis peptide bond. To test the kinetic model, we used simulations based on the six-state scheme and were able to completely reproduce the folding kinetics for Pro- SNase across a range of denaturant concentrations.