Roles of Hydration in Protein–Ligand Binding: Passive or Active Participant?

Hydration is a critical yet often underappreciated factor that influences protein dynamics in solution, with direct effects on structure, stability, and interactions such as ligand binding. Native mass spectrometry (nMS) enables the analysis of biomolecules in their solution states, which are shaped by cofactors, osmolytes, ligands, and notably, hydration. Here, we employ variable-temperature electrospray ionization to address a central question in molecular biophysics: does hydration act as a passive background solvent or as an active participant in modulating ligand binding? To investigate these effects, temperature-dependent changes in average charge state (Zavg), ADP equilibrium binding affinities (Ka), and enthalpy–entropy compensation (EEC) for the GroEL single ring mutant (SR1) were collected in both H2O and D2O. Temperature-dependent shifts in Zavg were observed for SR1-ADP complexes in both solvents, indicating protein conformational changes. Differences in nucleotide binding affinities calculated from mole fraction plots determined as a function of concentration between H2O and D2O solutions suggest that hydration plays a role in modulating ligand binding. Changes in hydration can modulate protein conformation and ligand binding affinities, typically reflected in shifts in enthalpy (ΔH) and entropy (−TΔS), while the overall Gibbs free energy (ΔG) remains relatively unchanged. Thermodynamic analysis revealed distinct patterns of EEC in D2O compared to H2O, providing insight into how hydration modulates the SR1(ADP)1–7 interactions. Collectively, these findings support the view that hydration acts as an active participant in ligand binding, with measurable effects on protein conformation, stability, and thermodynamics.