Centre de Biochimie Structurale, Institut National pour la Santé et la Recherche Médicale U554, Centre National pour la Recherche Scientifique Unité Mixte de Recherche 5048, Université Montpellier 1&2, Montpellier, France.
Proc Natl Acad Sci U S A. 2012 May 1;109(18):6945-50. doi: 10.1073/pnas.1200915109. Epub 2012 Apr 10.
It has been known for nearly 100 years that pressure unfolds proteins, yet the physical basis of this effect is not understood. Unfolding by pressure implies that the molar volume of the unfolded state of a protein is smaller than that of the folded state. This decrease in volume has been proposed to arise from differences between the density of bulk water and water associated with the protein, from pressure-dependent changes in the structure of bulk water, from the loss of internal cavities in the folded states of proteins, or from some combination of these three factors. Here, using 10 cavity-containing variants of staphylococcal nuclease, we demonstrate that pressure unfolds proteins primarily as a result of cavities that are present in the folded state and absent in the unfolded one. High-pressure NMR spectroscopy and simulations constrained by the NMR data were used to describe structural and energetic details of the folding landscape of staphylococcal nuclease that are usually inaccessible with existing experimental approaches using harsher denaturants. Besides solving a 100-year-old conundrum concerning the detailed structural origins of pressure unfolding of proteins, these studies illustrate the promise of pressure perturbation as a unique tool for examining the roles of packing, conformational fluctuations, and water penetration as determinants of solution properties of proteins, and for detecting folding intermediates and other structural details of protein-folding landscapes that are invisible to standard experimental approaches.
近 100 年来，人们已经知道压力会使蛋白质展开，但这种效应的物理基础尚不清楚。压力导致蛋白质展开意味着蛋白质展开状态的摩尔体积小于折叠状态的摩尔体积。这种体积的减少被认为是由于以下因素的差异：与蛋白质相关的、与体相水的密度不同的水的密度，体相水的结构随压力的变化，蛋白质折叠状态下内部空腔的丧失，或这三个因素的某种组合。在这里，我们使用 10 种含有空腔的葡萄球菌核酸酶变体，证明压力主要是通过折叠状态下存在而展开状态下不存在的空腔来使蛋白质展开的。高场 NMR 光谱和受 NMR 数据约束的模拟被用来描述葡萄球菌核酸酶折叠景观的结构和能量细节，这些细节通常无法通过使用更苛刻变性剂的现有实验方法来获得。除了解决一个关于蛋白质压力展开的详细结构起源的百年难题外，这些研究还说明了压力扰动作为一种独特工具的潜力，可用于研究包装、构象波动和水渗透作为蛋白质溶液性质决定因素的作用，以及用于检测折叠中间体和其他标准实验方法不可见的蛋白质折叠景观的结构细节。
