Terahertz Spectroscopy of Hydration Water in Crowded Protein Solutions

The dynamics of hydration water surrounding biomolecules play a crucial role in determining their structural stability, conformational flexibility, governing many of their interactions in the cellular environment. In crowded conditions, where high macromolecular concentrations prevail, water cannot be regarded as a passive solvent but instead acts as an active participant that mediates both intra and intermolecular processes. Although the subject has been the focus of several studies, a clear description of how hydration water is affected by the presence of biomolecules has not been achieved. This thesis explores the structural and dynamical properties of hydration water in highly concentrated aqueous protein solutions, by combining a variety of optical and terahertz spectroscopic techniques spanning complementary frequency and time domains. The first part of the work reports an Optical Kerr Effect spectroscopic study on water-lysozyme solutions at increasing concentrations. It was possible the direct separation of the contributions of bulk water, hydration water, and protein, revealing two distinct structural relaxation processes in the hydration shell. The faster process, occurring on a few-picosecond timescale, is associated with hydrogen-bond exchange dynamics, whereas the slower process, extending over tens of picoseconds, originates from water reorganization coupled to protein surface fluctuations. Two vibrational intermolecular modes of hydration water were also identified, corresponding to the bending and stretching vibrations of the hydrogen bond network. The amplitudes of both structural and vibrational components exhibit a marked discontinuity near the lysozyme concentration of 200–225 mg/mL, indicating a crossover in the hydration regime connected to the onset of protein clustering. Terahertz time-domain spectroscopy investigation were conducted on the same water-lysozyme samples, in attenuated total reflection geometry. Absorption coefficients and the refractive index were measured in the 0.1–3 THz frequency range at temperatures between 4 and 35 °C. The results show that at lower temperatures, the absorption coefficient of hydration water strongly depends on protein concentration. At higher temperatures, this dependence weakens, suggesting effects of enhanced instability and polydispersity of clusters. Measurements with a THz Fourier-transform infrared spectrometer were performed in the ZEMOS laboratory at Ruhr-Universität in Bochum (GER), enabled access to the full spectral range up to 18 THz. The analysis, based on the THz calorimetry framework, allowed the identification and quantification of two distinct hydration water populations: ’hydrophobic wrap’ water, characterized by a redshifted absorption band around 150 cm−1, and ’hydrophilic bound’ water, identified by the positive slope in the librational region (400–600 cm−1). Complementary Dynamic Light Scattering measurements confirmed the presence of clusters of varying size, whose growth correlates with the redistribution of water between these two populations. By mapping the variations in the corresponding spectroscopic observables, a specific THz phase diagram for the cluster regime was constructed, clearly differentiating it from the regimes associated with liquid–liquid phase separation and liquid–solid phase separation. Optical Pump–THz Probe spectroscopy was employed to investigate ultrafast energy transport in heme proteins (myoglobin and cytochrome c). This technique directly tracks the flow of vibrational energy from the optically excited heme group through the protein matrix to the surrounding solvent. By characterising the THz spectrum of hydration water, it was possible to obtain the relaxation times, ranging from 6 to 10 ps and showing a clear isotope effect between H2O and D2O, are in excellent agreement with diffusive energy transfer models. These results reveal that thermal energy dissipation occurs predominantly through vibrational diffusion within the protein matrix, followed by coupling to the hydration shell, which strongly influences the characteristic time of this process. Overall, this study provides new insights into how hydration water reorganizes and mediates the collective behaviour of proteins. The results contribute to the understanding of fundamental mechanisms governing biomolecular hydration, clustering, and phase transitions, and establish terahertz spectroscopy as a powerful tool for probing complex aqueous biological systems.