Mosaic of Surface Interaction Motifs Governs Hydration-Dependent Water Dynamics in Lysozyme

Abstract Hydration-dependent dynamics of interfacial water in proteins are often interpreted as arising from the gradual evolution of a continuous hydration layer, yet the microscopic origin of this behavior remains unclear. Here, we investigate whether hydration-dependent dynamics of interfacial water in lysozyme powders arise from continuous hydration-layer evolution or from redistribution among discrete surface environments. Interfacial water residence times, computed using a continuous occupancy criterion across the full temperature range of 248–283 K and hydration levels of 60–250 wt %, follow Arrhenius behavior under all conditions studied. Apparent activation energies, extracted from linear fits of ln τ versus 1/T, vary modestly between approximately 6.5 and 8.0 kJ mol–1 across hydration levels. Residue-resolved analysis reveals pronounced spatial heterogeneity in interfacial hydration, intermolecular contacts, and hydrogen bonding across the protein surface. Clustering of residue-level fingerprints identifies four distinct surface interaction motifs (hydration-dominated, contact-stabilized, hydrogen-bond-enriched, and weakly interacting) whose relative populations shift systematically but modestly with both hydration and temperature. The modest hydration dependence of apparent activation energies is consistent with thermodynamic reweighting among these motifs, rather than requiring uniform evolution of a single hydration layer, though an additional contribution from a hydration-threshold transition cannot be excluded.