PROBING HYDRATION CONTRIBUTIONS TO THE THERMODYNAMICS OF LIGAND-BINDING BY PROTEINS - ENTHALPY AND HEAT-CAPACITY CHANGES OF TACROLIMUS AND RAPAMYCIN BINDING TO FK506 BINDING-PROTEIN IN D2O AND H2O

The stabilities of native proteins and protein-ligand complexes result from differential interactions among numerous polar and nonpolar atom s within the proteins and ligands and of these atoms with water. Delin eation of the various energetic contributions of the stabilities of pr oteins or protein-ligand complexes in aqueous solution, and an evaluat ion of their structural basis, requires a direct account of the change s, in the interactions of the protein with the solvent, that accompany the folding or binding reactions. Two largely nonpolar, structurally related macrolide ligands, tacrolimus (also known as FK506) and rapamy cin, each bind with high affinity to a common site on a small FK506 bi nding protein (FKBP-12) and inhibit its peptidylprolyl cis-trans-isome rase activity. In an effort to elucidate the influence of water on the thermodynamics of their binding reactions, we have measured the entha lpies of tacrolimus and rapamycin binding to FKBP-12, in buffered solu tions of H2O (at pH 7.0) or D2O (at pD 7.0), by high-precision titrati on calorimetry in the temperature range 5-30-degrees-C. For both tacro limus and rapamycin binding, a large enthalpic destabilization of bind ing is observed in D2O relative to H2O, in the temperature range exami ned. Additionally, large negative constant pressure heat capacity chan ges are observed for the binding of the ligands in both H2O and D2O. A thermodynamic analysis is presented to identify the structural determ inants of the differences in the energetics of binding in light and he avy water. The analysis suggests that a chief contributor to the obser ved enthalpic destabilization is the differential hydration, of protei n and ligand atoms, by light and heavy water.