{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,10,2]],"date-time":"2026-10-02T03:59:08Z","timestamp":1790913548724,"version":"4.1.0"},"posted":{"date-parts":[[2026,9,30]]},"group-title":"Biochemistry","reference-count":76,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T00:00:00Z","timestamp":1790726400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"award":["RM1GM149374"],"award-info":[{"award-number":["RM1GM149374"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["RM1GM145416"],"award-info":[{"award-number":["RM1GM145416"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R01GM134063-01"],"award-info":[{"award-number":["R01GM134063-01"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["A-2162"],"award-info":[{"award-number":["A-2162"]}],"id":[{"id":"https:\/\/ror.org\/00np6vq88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["A-2106"],"award-info":[{"award-number":["A-2106"]}],"id":[{"id":"https:\/\/ror.org\/00np6vq88","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,9,30]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  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 \u00b0C), enabling determination of binding affinities and the associated Gibbs free energy, enthalpy, entropy, heat capacity changes (\u0394C\n                  <jats:sub>p<\/jats:sub>\n                  ), and enthalpy-entropy compensation (EEC) for each binding reaction. Temperature-dependent changes in van\u2019t Hoff curvature produce distinct \u0394C\n                  <jats:sub>p<\/jats:sub>\n                  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 \u223c23 \u00b0C, 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\n                  <jats:sub>2<\/jats:sub>\n                  O and D\n                  <jats:sub>2<\/jats:sub>\n                  O reveals pronounced differences in van\u2019t Hoff curvature, \u0394C\n                  <jats:sub>p<\/jats:sub>\n                  , 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.\n                <\/jats:p>","DOI":"10.64898\/2026.09.29.755381","type":"posted-content","created":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T12:15:19Z","timestamp":1790770519000},"source":"Crossref","is-referenced-by-count":0,"title":["Beyond Structure: Protein and Solution Dynamics Shape Ligand-Binding Thermodynamics"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-9040-6750","authenticated-orcid":false,"given":"Kacie A.","family":"Evans","sequence":"first","affiliation":[{"name":"Department of Chemistry, Texas A&M University, College Station, Texas 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