Université Lille Nord de France, Lille, France.
J Phys Chem B. 2011 May 26;115(20):6740-8. doi: 10.1021/jp2014836. Epub 2011 May 4.
Pressure denaturation of lysozyme dissolved in H(2)O and D(2)O was analyzed using Raman investigations in a wide frequency range. The simultaneous analysis of regions corresponding to the molecular fingerprint of the protein (500-1800 cm(-1)), and the low- (50-450 cm(-1)) and high- (2600-3800 cm(-1)) frequency spectra, allow us to probe protein denaturation and the organization of water molecules. The pressure- and heat-induced transformations are compared. Both pressure- and heat-denatured states are obtained through an intermediate state characterized by intact secondary structure and enhanced water penetration in the tertiary structure. As a consequence of a weaker penetration upon pressurizing, it was found that the pressure-denatured state was partially unfolded compared with the heat-denatured state. The mechanism of pressure denaturation was related to the disruption of the hydrogen-bond network of water onto a set of clusters characterized by strengthened O - H interactions, inducing a hardening of protein dynamics. The mechanism is opposite to that observed upon heating, i.e., the softening of the hydrogen bond network of water inducing a softer protein dynamics. The analysis of the intramolecular O-H stretching reveals that pressurizing lysozyme aqueous solution favors the development of low-density water from the protein surface to the bulk, contrasting to the compression of pure water leading to crystallization of high-density ice-VI.
采用喇曼光谱法在较宽的频率范围内研究了溶解在 H(2)O 和 D(2)O 中的溶菌酶的压力变性。同时分析了对应于蛋白质分子指纹的区域（500-1800 cm(-1)）、低频（50-450 cm(-1)）和高频（2600-3800 cm(-1)）光谱，从而可以探测蛋白质变性和水分子的组织。比较了压力和热诱导的转变。通过中间状态得到压力和热变性状态，该中间状态的特征为完整的二级结构和三级结构中增强的水分子渗透。由于加压时渗透较弱，发现与热变性状态相比，压力变性状态部分展开。压力变性的机制与水的氢键网络破坏有关，水的氢键网络破坏到一组以强化 O-H 相互作用为特征的簇上，从而导致蛋白质动力学变硬。该机制与加热时观察到的相反，即水的氢键网络软化导致蛋白质动力学变软。对分子内 O-H 伸缩的分析表明，加压溶菌酶水溶液有利于从蛋白质表面到体相形成低密度水，这与导致高密度冰-VI 结晶的纯水压缩相反。
