Site Specific Hydration Contributes to the Thermal Stability of Proteins and Protein-Complexes

Water is integral to protein structure and stability, yet ensemble measurements rarely identify the hydration interactions associated with stabilization. Here, variable-temperature electrospray ionization ion mobility mass spectrometry, conformation-resolved thermodynamic analysis, and molecular dynamics simulations are combined to examine solvent-dependent thermal stability of chymotrypsin inhibitor 2 (CI-2), a model two-state folder, and the barnase-barstar (Bn-B*) complex, which contains a highly polar binding interface. Replacing H ₂ O with D ₂ O raises the thermal transition temperature of both systems by approximately 3 °C, favors compact conformations, and reduces the relative thermodynamic accessibility of extended states. The conformationally resolved enthalpy-entropy compensation reveals D ₂ O increases enthalpy for CI-2 but reduces enthalpy for Bn-B* as a result of opposing effects on the individual subunits of the complex. Molecular dynamics simulations reveal that lower solvent-accessible surface areas and fewer protein-water hydrogen bonds in D ₂ O coexist with longer water residence times at specific structural sites. In CI-2, the strongest changes localize to the second β-strand/C-terminal region; in Bn-B*, persistent hydration is concentrated at the charged protein-protein interface. Together, these results support a model in which D ₂ O dependent stabilization is associated not only with the extent of hydration, but the site and lifetime of water interactions. More broadly, this work provides an experimental framework for resolving solvent-dependent conformational thermodynamics of protein unfolding and interactions, enabling direct comparison of free-energy landscapes across solution environments.