Luigi Caminiti

Lysozyme is a well-known globular protein that can form self-assembled clusters. Here, we report the results of a study on the changes in solvation dynamics upon increasing protein concentration via THz spectroscopy. We find spectral solvation fingerprints in the THz frequency range, which are clearly distinguished from those for liquid–liquid phase separation or liquid–solid phase separation. Furthermore, we carried out supplementary measurements using dynamic light scattering to characterize the size distribution of the protein clusters. This study sheds light on using THz studies of solvation dynamics to characterize and differentiate protein hydration in the case of protein assembly, liquid–liquid phase separation, and liquid–solid phase separation.

Optically excited heme proteins release their energy into the hydration water in approximately 7 ps, subsequent to internal vibrational redistribution. Adapted from an image created by S.Duce.

We propose a time-resolved optical Kerr effect study of the structural and vibrational dynamics of the hydration water surrounding the lysozyme on a very fast time scale. Measurements as a function of lysozyme concentration make it possible to distinguish the hydration water contribution from that of both the bulk water and the protein. Our results provide experimental evidence of the existence of two structural dynamics of hydration water, associated with a hydrogen bond exchange relaxation process and with the reorganization of water molecules induced by protein structural fluctuations. Likewise, we evaluated the vibrational dynamics of the water hydration layer at subpicosecond time scales. Our measurements of hydration water properties reveal the presence of a crossover point at a specific protein concentration. This crossover marks the transition between two clustering regimes with distinct hydration characteristics and establishes a possible threshold for protein crowding.

Water is not only a solvent in which biological molecules are dissolved, but it also plays an essential and active role in the proper development of biological processes within living organism. Water hydration shell surrounds biomolecules and has a plasticizing impact on their backbone, allowing them to execute their physiological activity in biochemical processes and biophysical modulations. Although the essential role of water in biology is evident, the comprehension of hydration water properties remains limited; the mutual influence between the protein surface and the hydration layers, on regulating structure and dynamics, remains a strongly debated question. In particular, the experimental and simulation findings on fast dynamics of hydration water are still controversial. We propose a time-resolved optical Kerr effect study of the structural and vibrational dynamics of the hydration water surrounding lysozyme on a very fast time scale. Measurements as a function of lysozyme concentration makes it possible to distinguish the hydration water contribution from that of both the bulk water and the protein. Our results provide the experimental evidence of the existence of two structural dynamics of hydration water, associated respectively with a hydrogen bond exchange relaxation process and with the reorganization of water molecules induced by protein structural fluctuations. Likewise, we evaluated the vibrational dynamics of the water hydration layer at sub-picosecond time scales. Our study of the dynamics of hydration water reveals a lysozyme clustering phenomenon prompted by the self-crowding environment, which is associated with the liquid-liquid phase separation mechanisms inherent in these protein solutions.