Nucci Nathaniel V, Fuglestad Brian, Athanasoula Evangelia A, Wand A Joshua
Department of Biochemistry and Biophysics and Johnson Research Foundation, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104.
Department of Biochemistry and Biophysics and Johnson Research Foundation, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA 19104
Proc Natl Acad Sci U S A. 2014 Sep 23;111(38):13846-51. doi: 10.1073/pnas.1410655111. Epub 2014 Sep 8.
It is well known that high hydrostatic pressures can induce the unfolding of proteins. The physical underpinnings of this phenomenon have been investigated extensively but remain controversial. Changes in solvation energetics have been commonly proposed as a driving force for pressure-induced unfolding. Recently, the elimination of void volumes in the native folded state has been argued to be the principal determinant. Here we use the cavity-containing L99A mutant of T4 lysozyme to examine the pressure-induced destabilization of this multidomain protein by using solution NMR spectroscopy. The cavity-containing C-terminal domain completely unfolds at moderate pressures, whereas the N-terminal domain remains largely structured to pressures as high as 2.5 kbar. The sensitivity to pressure is suppressed by the binding of benzene to the hydrophobic cavity. These results contrast to the pseudo-WT protein, which has a residual cavity volume very similar to that of the L99A-benzene complex but shows extensive subglobal reorganizations with pressure. Encapsulation of the L99A mutant in the aqueous nanoscale core of a reverse micelle is used to examine the hydration of the hydrophobic cavity. The confined space effect of encapsulation suppresses the pressure-induced unfolding transition and allows observation of the filling of the cavity with water at elevated pressures. This indicates that hydration of the hydrophobic cavity is more energetically unfavorable than global unfolding. Overall, these observations point to a range of cooperativity and energetics within the T4 lysozyme molecule and illuminate the fact that small changes in physical parameters can significantly alter the pressure sensitivity of proteins.
众所周知，高静水压力可诱导蛋白质展开。这一现象的物理基础已得到广泛研究，但仍存在争议。溶剂化能的变化通常被认为是压力诱导展开的驱动力。最近，有人认为消除天然折叠状态下的空穴体积是主要决定因素。在这里，我们使用含空腔的T4溶菌酶L99A突变体，通过溶液核磁共振光谱研究这种多结构域蛋白质的压力诱导去稳定化。含空腔的C末端结构域在中等压力下完全展开，而N末端结构域在高达2.5千巴的压力下基本保持结构完整。苯与疏水空腔的结合抑制了对压力的敏感性。这些结果与假野生型蛋白质形成对比，假野生型蛋白质的残余空穴体积与L99A-苯复合物非常相似，但在压力下显示出广泛的亚全局重组。将L99A突变体封装在反胶束的水纳米级核心中，用于研究疏水空腔的水合作用。封装的受限空间效应抑制了压力诱导的展开转变，并使得在高压下观察到空腔被水填充。这表明疏水空腔的水合作用在能量上比全局展开更不利。总体而言，这些观察结果表明T4溶菌酶分子内存在一系列协同性和能量关系，并阐明了物理参数的微小变化可显著改变蛋白质压力敏感性这一事实。
