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

## TL;DR

This study shows that hydration actively influences how proteins bind to ligands, affecting their structure and stability.

## Contribution

The paper demonstrates hydration's active role in ligand binding through thermodynamic and conformational analysis using native mass spectrometry.

## Key findings

- Hydration modulates ligand binding affinities and protein conformation.
- Temperature-dependent shifts in charge states indicate structural changes.
- Distinct enthalpy-entropy compensation patterns in D2O versus H2O reveal hydration's thermodynamic impact.

## Abstract

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 (Z
avg),
ADP equilibrium binding affinities (K
a), and enthalpy–entropy compensation (EEC) for the GroEL single
ring mutant (SR1) were collected in both H2O and D2O. Temperature-dependent shifts in Z
avg 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.