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标题：Role of cavities and hydration in the pressure unfolding of T4 lysozyme
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作者：Nathaniel V. Nucci ; Brian Fuglestad ; Evangelia A. Athanasoula 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2014
卷号：111
期号：38
页码：13846-13851
DOI：10.1073/pnas.1410655111
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificancePressure unfolding of proteins is a fundamental aspect of their thermodynamic response, the origins of which remain controversial. Here, we use high-pressure solution NMR to investigate the pressure response of a model protein, T4 lysozyme, under various conditions. Our data resolve longstanding controversies regarding the pressure response of this protein and the hydration of the internal hydrophobic cavity. It is shown that local packing (cavities) and the availability of conformational space have important and nonlocal impacts on the protein pressure response. Overall, the findings presented here reveal a previously unappreciated complexity in the pressure response of protein structure. 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.
关键词：protein stability ; protein folding and cooperativity ; protein hydration ; high-pressure NMR ; reverse micelle encapsulation