Biology subjects

Rider, R. L.

Publications and source records attributed to Rider, R. L..

2 recordsLinked to original sources

bioRxiv2026

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 H2O with D2O raises the thermal transition temperature of both systems by approximately 3 {degrees}C, favors compact conformations, and reduces the relative thermodynamic accessibility of extended states. The conformationally resolved enthalpy-entropy compensation reveals D2O 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 D2O coexist with longer water residence times at specific structural sites. In CI-2, the strongest changes localize to the second {beta}-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 D2O 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.

biochemistry↗

bioRxiv2026

Beyond Structure: Protein and Solution Dynamics Shape Ligand-BindingThermodynamics

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 {degrees}C), enabling determination of binding affinities and the associated Gibbs free energy, enthalpy, entropy, heat capacity changes ({Delta}Cp), and enthalpy-entropy compensation (EEC) for each binding reaction. Temperature-dependent changes in van,t Hoff curvature produce distinct {Delta}Cp 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 {degrees}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 H2O and D2O reveals pronounced differences in van,t Hoff curvature,{Delta}Cp, 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.

biochemistry↗