Analysis of the mechanism of lysozyme pressure denaturation from Raman spectroscopy investigations, and comparison with thermal denaturation.

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.