Hierarchical folding mechanism of apomyoglobin revealed by ultra-fast H/D exchange coupled with 2D NMR
Hierarchical folding mechanism of apomyoglobin revealed by ultra-fast H/D exchange coupled with 2D NMR
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DOI：
10.1073/pnas.0804033105
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发表时间：
2008-09-16
影响因子：
11.1
通讯作者：
Wright, Peter E.
中科院分区：
文献类型：
--
作者：
Uzawa, Takanori;Nishimura, Chiaki;Wright, Peter E.
蛋白质折叠的最早步骤是在极快的时间尺度上完成的，这是很难通过实验获得的。我们使用快速混合猝灭流动方法扩展了脱脂蛋白折叠研究的时间分辨率，并阐明了在这一过程中在亚毫秒时间尺度上填充的中间态集合成员的结构和动力学特征。出现的画面是一个快速相互转换的状态的连续体。即使在仅0.4毫秒的复性时间之后，也会形成一种致密的状态，其中包含A、G和H螺旋的主要部分，这些螺旋被折叠得足够好，以保护酰胺不被交换。在该核心上寻找对接位置时，B、C和E螺旋区域折叠较慢，在打开和关闭状态之间波动较快；随着重折叠时间从0.4ms增加到6ms，这些区域的二级结构变得稳定。在折叠时间为6ms时，A、G、H芯没有进一步的稳定。这些研究开始对完全展开状态和自然折叠状态之间的紧凑状态的级数进行时间解析，并确认当蛋白质在其折叠轨迹上搜索构象空间时，存在以分层序列相互转换的中间体集合。
The earliest steps in the folding of proteins are complete on an extremely rapid time scale that is difficult to access experimentally. We have used rapid-mixing quench-flow methods to extend the time resolution of folding studies on apomyoglobin and elucidate the structural and dynamic features of members of the ensemble of intermediate states that are populated on a submillisecond time scale during this process. The picture that emerges is of a continuum of rapidly interconverting states. Even after only 0.4 ms of refolding time a compact state is formed that contains major parts of the A, G, and H helices, which are sufficiently well folded to protect amides from exchange. The B, C, and E helix regions fold more slowly and fluctuate rapidly between open and closed states as they search docking sites on this core; the secondary structure in these regions becomes stabilized as the refolding time is increased from 0.4 to 6 ms. No further stabilization occurs in the A, G, H core at 6 ms of folding time. These studies begin to time-resolve a progression of compact states between the fully unfolded and native folded states and confirm the presence an ensemble of intermediates that interconvert in a hierarchical sequence as the protein searches conformational space on its folding trajectory.