Site-specific water dynamics drives protein stability in hydrated deep eutectic solvents.

Deep eutectic solvents (DESs) have gained prominence as promising biocatalytic media owing to their tunability, cost-effectiveness, and biocompatibility. However, a comprehensive mechanistic understanding of protein behavior in such environments remains elusive. In this study, we explore the role of associated water dynamics in modulating the thermal stability of human serum albumin within hydrated choline chloride-glycerol and choline chloride-ethylene glycol DESs. By employing domain-specific site-selective fluorescence labeling and red-edge excitation shift measurement, alongside circular dichroism spectroscopy, we reveal that modulation of associated water dynamics distinctly alters the entropic and enthalpic contributions to the protein stability. Flexible associated water enhances enthalpic stabilization but promotes entropic destabilization, whereas restricted associated water induces the opposite effect, providing a mechanistic basis for the entropy-enthalpy compensation. Our findings underscore the necessity of domain-specific stability measurements in multidomain proteins and highlight the pivotal role of immediate hydration dynamics in protein stabilization within unconventional solvent systems. This study offers valuable insights for the rational design of DES-based biocatalytic systems.