Site-specific hydration dynamics in the nonpolar core of a molten globule by dynamic nuclear polarization of water.
Site-specific hydration dynamics in the nonpolar core of a molten globule by dynamic nuclear polarization of water.
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DOI：
10.1021/ja111515s
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发表时间：
2011-04-20
影响因子：
15
通讯作者：
Han, Songi
中科院分区：
文献类型：
--
作者：
Armstrong, Brandon D.;Choi, Jennifer;Lopez, Carlos;Wesener, Darryl A.;Hubbell, Wayne;Cavagnero, Silvia;Han, Songi
水-蛋白质相互作用在蛋白质折叠中起着直接的作用。伴随蛋白质折叠的链崩溃涉及水从非极性核心的挤出。对于许多蛋白质，包括脱辅基肌红蛋白（apoMb），疏水相互作用驱动初始折叠到中间状态，然后折叠到最终结构。然而，关于坍缩的中间状态的核心是否是水合的，如果是的话，这种水的动态性质是什么，争论仍在继续。一个关键的挑战是，蛋白质水化动力学是显着异质性，但缺乏合适的实验技术，用于测量水化动力学与位点特异性。在这里，我们介绍Overhauser动态核极化在0.35 T通过位点特异性氮氧自由基自旋标签作为一种独特的工具，探测内部和表面蛋白质水合动力学与特定位点的分辨率在熔融球状，原生和未折叠的蛋白质状态。水的1H NMR信号增强携带关于在自旋标记的~10 μ m内的溶剂的局部动力学的信息。EPR被协同使用，以获得对自旋标记蛋白质的局部极性和流动性的见解。几个埋和溶剂暴露的网站apoMb进行检查，每个轴承共价结合的氮氧自旋标签。我们发现，疏水核心的apoMb熔融球水合水轴承显着的平移动力学，只有4-6倍慢于散装水。天然状态的水化动力学是不均匀的，而酸展开状态承载快速扩散的水化水。这项研究提供了一个高分辨率的蛋白质水合动力学的折叠依赖性一瞥。
Water-protein interactions play a direct role in protein folding. The chain collapse that accompanies protein folding involves extrusion of water from the nonpolar core. For many proteins, including apomyoglobin (apoMb), hydrophobic interactions drive an initial collapse to an intermediate state before folding to the final structure. However, the debate continues as to whether the core of the collapsed intermediate state is hydrated and, if so, what the dynamic nature of this water is. A key challenge is that protein hydration dynamics is significantly heterogeneous, yet suitable experimental techniques for measuring hydration dynamics with site-specificity are lacking. Here, we introduce Overhauser dynamic nuclear polarization at 0.35 T via site-specific nitroxide spin labels as a unique tool to probe internal and surface protein hydration dynamics with site-specific resolution in the molten globular, native, and unfolded protein states. The 1H NMR signal enhancement of water carries information about the local dynamics of the solvent within ~10 Å of a spin label. EPR is used synergistically to gain insights on local polarity and mobility of the spin-labeled protein. Several buried and solvent-exposed sites of apoMb are examined, each bearing a covalently bound nitroxide spin label. We find that the hydrophobic core of the apoMb molten globule is hydrated with water bearing significant translational dynamics, only 4–6-fold slower than that of bulk water. The hydration dynamics of the native state is heterogeneous, while the acid-unfolded state bears fast-diffusing hydration water. This study provides a high-resolution glimpse at the folding-dependent nature of protein hydration dynamics.
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