In the present study the authors examine the thermodn. of binding of two related pyrazine-derived ligands to the major urinary protein, MUP-I, using a combination of isothermal titrn. calorimetry (ITC), x-ray crystallog., and NMR backbone 15N and Me side-chain 2H relaxation measurements. Global thermodn. data derived from ITC indicate that binding is driven by favorable enthalpic contributions, rather than the classical entropy-driven hydrophobic effect. Unfavorable entropic contributions from the protein backbone and side-chain residues in the vicinity of the binding pocket are partially offset by favorable entropic contributions at adjacent positions, suggesting a "conformational relay" mechanism whereby increased rigidity of residues on ligand binding are accompanied by increased conformational freedom of side chains in adjacent positions. The principal driving force governing ligand affinity and specificity can be attributed to solvent-driven enthalpic effects from desolvation of the protein binding pocket. [on SciFinder (R)]