title: A large solvent isotope effect on protein association thermodynamics.
abstract: Solvent reorganization can contribute significantly to the energetics of protein-protein interactions. However, our knowledge of the magnitude of the energetic contribution is limited, in part, by a dearth of quantitative experimental measurements. The biotin repressor forms a homodimer as a prerequisite to DNA binding to repress transcription initiation. At 20 °C, the dimerization reaction, which is thermodynamically coupled to binding of a small ligand, bio-5'-AMP, is characterized by a Gibbs free energy of -7 kcal/mol. This modest net dimerization free energy reflects underlying, very large opposing enthalpic and entropic driving forces of 41 ± 3 and -48 ± 3 kcal/mol, respectively. The thermodynamics have been interpreted as indicating coupling of solvent release to dimerization. In this work, this interpretation has been investigated by measuring the effect of replacing H2O with D2O on the dimerization thermodynamics. Sedimentation equilibrium measurements performed at 20 °C reveal a solvent isotope effect of -1.5 kcal/mol on the Gibbs free energy of dimerization. Analysis of the temperature dependence of the reaction in D2O indicates enthalpic and entropic contributions of 28 and -37 kcal/mol, respectively, considerably smaller than the values measured in H2O. These large solvent isotope perturbations to the thermodynamics are consistent with a significant contribution of solvent release to the dimerization reaction.
authors.affiliation: Department of Chemistry and Biochemistry, College of Computer, Mathematical and Natural Sciences, University of Maryland , College Park, Maryland 20742, United States.
authors.forename: Christopher
authors.initials: C
authors.lastname: Eginton
authors.forename: Dorothy
authors.initials: D
authors.lastname: Beckett
mesh_terms: D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000737:chemistry; D001710:Biotin / Q000031:analogs & derivatives / Q000737:chemistry; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry*; D017666:Deuterium Oxide / Q000737:chemistry / Q000494:pharmacology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D011485:Protein Binding; D055503:Protein Multimerization / Q000187:drug effects; D012097:Repressor Proteins / Q000737:chemistry*; D012997:Solvents / Q000494:pharmacology*; D013816:Thermodynamics; D014867:Water / Q000737:chemistry
chemical_list: C082099:5'-AMP-biotin; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; D012997:Solvents; D014867:Water; D000249:Adenosine Monophosphate; D001710:Biotin; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli; D017666:Deuterium Oxide