Abstract
Abstract
Variable-temperature electrospray ionization native mass spectrometry (vT-ESI nMS) provides a means to determine how temperature-dependent changes in protein and solution dynamics influence individual ligand-binding reactions within multiligand protein complexes. Here, the GroEL single-ring mutant (SR1), which binds up to seven nucleotides, serves as a model system due to its sensitivity to solution conditions. Native MS resolves individual ligand-bound populations, while vT-ESI extends these measurements across temperature (3-43 °C), enabling determination of binding affinities and the associated Gibbs free energy, enthalpy, entropy, heat capacity changes (ΔC
p
), and enthalpy-entropy compensation (EEC) for each binding reaction. Temperature-dependent changes in van’t Hoff curvature produce distinct ΔC
p
profiles, indicating changes in the dynamics governing ligand binding that may reflect contributions from protein conformational and protonation microstates. Temperature-dependent shifts in average charge state, consistent with changes in solvent-accessible surface area, indicate corresponding changes in protein conformation. Binding is predominantly enthalpy-driven below ∼23 °C, with increasing entropic contributions at higher temperatures, while the seventh ADP binding reaction exhibits a distinct EEC profile. Comparison of the seventh ADP binding reaction in H
2
O and D
2
O reveals pronounced differences in van’t Hoff curvature, ΔC
p
, and EEC, demonstrating that changes in the hydration environment alter the thermodynamic response. Collectively, these measurements show that temperature-dependent changes in protein and solution dynamics alter the distributions and thermodynamics of individual ligand-bound states and demonstrate the utility of vT-ESI nMS for resolving these effects in complex multiligand systems.