An Overlapping Region between the Two Terminal Folding Units of the Outer Surface Protein A (OspA) Controls Its Folding Behavior
An Overlapping Region between the Two Terminal Folding Units of the Outer Surface Protein A (OspA) Controls Its Folding Behavior
复制标题
外表面蛋白 A (OspA) 两个末端折叠单元之间的重叠区域控制其折叠行为
DOI：
10.1016/j.jmb.2018.04.025
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发表时间：
2018
影响因子：
5.6
通讯作者：
Kuwajima Kunihiro
中科院分区：
文献类型：
--
作者：
Makabe Koki;Nakamura Takashi;Dhar Debanjan;Ikura Teikichi;Koide Shohei;Kuwajima Kunihiro
虽然许多自然存在的蛋白质由多个结构域组成，但迄今为止对蛋白质折叠的研究大多是针对单结构域蛋白质或多结构域蛋白质的孤立结构域。为了进一步加深我们对蛋白质折叠机制的理解，需要对多结构域蛋白质折叠进行研究。疏螺旋体外表面蛋白A(OspA)是一种富含β的双结构域蛋白，它的两个球状结构域由一个坚硬而稳定的单层β片层连接。因此，OspA特别适合作为研究蛋白质折叠中结构域之间相互作用的模型系统。在这里，我们用色氨酸荧光和紫外圆二色谱研究了尿素诱导的OspA折叠-去折叠反应的平衡和动力学。对实验数据的整体分析揭示了令人信服的证据，表明在动力学重折叠过程中积累了路径上的中间体，并证明了动力学中间体与先前描述的平衡展开中间体之间的一致性。结果表明，该中间体在N-末端结构域和单层β-Sheet中具有完全天然的结构，C-末端结构域仍未折叠。对产生性路径上折叠中间体的观察清楚地表明，在单层β-Sheet介导的两个结构域之间存在着实质性的相互作用。我们认为，两个结构域之间的刚性和稳定的中间区域在两个折叠单元之间产生重叠，并可以能量耦合它们的折叠反应。
Although many naturally occurring proteins consist of multiple domains, most studies on protein folding to date deal with single-domain proteins or isolated domains of multi-domain proteins. Studies of multi-domain protein folding are required for further advancing our understanding of protein folding mechanisms. Borrelia outer surface protein A (OspA) is a β-rich two-domain protein, in which two globular domains are connected by a rigid and stable single-layer β-sheet. Thus, OspA is particularly suited as a model system for studying the interplays of domains in protein folding. Here, we studied the equilibria and kinetics of the urea-induced folding–unfolding reactions of OspA probed with tryptophan fluorescence and ultraviolet circular dichroism. Global analysis of the experimental data revealed compelling lines of evidence for accumulation of an on-pathway intermediate during kinetic refolding and for the identity between the kinetic intermediate and a previously described equilibrium unfolding intermediate. The results suggest that the intermediate has the fully native structure in the N-terminal domain and the single layer β-sheet, with the C-terminal domain still unfolded. The observation of the productive on-pathway folding intermediate clearly indicates substantial interactions between the two domains mediated by the single-layer β-sheet. We propose that a rigid and stable intervening region between two domains creates an overlap between two folding units and can energetically couple their folding reactions.