Probing the lysozyme hydration shell during thermal denaturation via FTIR spectroscopy.

This study employs Fourier-transform infrared (FTIR) spectroscopy combined with the HDO difference spectra method to investigate the evolution of the lysozyme hydration shell during thermal denaturation (25-85 °C). The hydration process involves two distinct regimes: a pre-denaturation stage (25-65 °C) and the subsequent thermal denaturation phase (75-85 °C). Across the entire temperature range, the hydration shell maintains remarkable thermal stability compared to bulk water. Crucially, hydration water actively responds to the protein's dynamic structural changes. At 65 °C, it senses the microscopic onset of thermal denaturation. Driven by a transient loss of structural rigidity, the protein exposes previously buried hydrophobic groups while preserving its native fold. This sustains water hydrogen-bond cooperativity, generating an enhanced hydration layer with the highest structural heterogeneity, and a local maximum in its structural entropy. An isothermal comparison between the hydration shells of the native and denatured states at 85 °C reveals that the denaturation-induced spatial separation of polar and non-polar groups partially disrupts this cooperativity. This leads to a weakening of the water hydrogen-bond network compared to the native state. Our findings demonstrate that hydration water acts as a sensitive molecular probe, reflecting the earliest stages of protein denaturation before macroscopic unfolding occurs.