Howard P Isermann Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA.
Proteins. 2010 May 15;78(7):1641-51. doi: 10.1002/prot.22680.
Many globular proteins unfold when subjected to several kilobars of hydrostatic pressure. This "unfolding-up-on-squeezing" is counter-intuitive in that one expects mechanical compression of proteins with increasing pressure. Molecular simulations have the potential to provide fundamental understanding of pressure effects on proteins. However, the slow kinetics of unfolding, especially at high pressures, eliminates the possibility of its direct observation by molecular dynamics (MD) simulations. Motivated by experimental results-that pressure denatured states are water-swollen, and theoretical results-that water transfer into hydrophobic contacts becomes favorable with increasing pressure, we employ a water insertion method to generate unfolded states of the protein Staphylococcal Nuclease (Snase). Structural characteristics of these unfolded states-their water-swollen nature, retention of secondary structure, and overall compactness-mimic those observed in experiments. Using conformations of folded and unfolded states, we calculate their partial molar volumes in MD simulations and estimate the pressure-dependent free energy of unfolding. The volume of unfolding of Snase is negative (approximately -60 mL/mol at 1 bar) and is relatively insensitive to pressure, leading to its unfolding in the pressure range of 1500-2000 bars. Interestingly, once the protein is sufficiently water swollen, the partial molar volume of the protein appears to be insensitive to further conformational expansion or unfolding. Specifically, water-swollen structures with relatively low radii of gyration have partial molar volume that are similar to that of significantly more unfolded states. We find that the compressibility change on unfolding is negligible, consistent with experiments. We also analyze hydration shell fluctuations to comment on the hydration contributions to protein compressibility. Our study demonstrates the utility of molecular simulations in estimating volumetric properties and pressure stability of proteins, and can be potentially extended for applications to protein complexes and assemblies.
许多球状蛋白质在受到几千巴静水压力时会展开。这种“受压展开”与人们的预期相反，因为人们预计随着压力的增加，蛋白质会受到机械压缩。分子模拟有可能为压力对蛋白质的影响提供基本的理解。然而，展开的缓慢动力学，特别是在高压下，排除了通过分子动力学（MD）模拟直接观察它的可能性。受实验结果的启发——压力变性状态是水膨胀的，以及理论结果——随着压力的增加，水向疏水接触转移变得有利，我们采用水插入法生成蛋白质葡萄球菌核酸酶（Snase）的展开状态。这些展开状态的结构特征——它们的水膨胀性质、保留的二级结构和整体紧凑性——模拟了实验中观察到的特征。使用折叠和展开状态的构象，我们在 MD 模拟中计算它们的偏摩尔体积，并估计压力依赖性的展开自由能。Snase 的展开体积为负（在 1 巴时约为-60 mL/mol），并且对压力相对不敏感，导致其在 1500-2000 巴的压力范围内展开。有趣的是，一旦蛋白质充分水膨胀，蛋白质的偏摩尔体积似乎对进一步的构象扩展或展开不敏感。具体来说，具有相对低回转半径的水膨胀结构的偏摩尔体积与明显更展开的状态相似。我们发现展开时的压缩性变化可以忽略不计，与实验结果一致。我们还分析了水合壳波动，以评论水合对蛋白质压缩性的贡献。我们的研究表明分子模拟在估计蛋白质的体积性质和压力稳定性方面的实用性，并可潜在地扩展到蛋白质复合物和组装体的应用。
