Role of water in protein-ligand interactions: Volumetric characterization of the binding of 2 '-CMP and 3 '-CMP to ribonuclease A

We report the first characterization of ligand-protein binding performed by a combination of volumetric, spectroscopic, and high-pressure measurements . We have used ultrasonic velocimetry, high-precision densimetry, and UV ab sorbance and CD spectroscopy in conjunction with high-pressure UV melting t o detect and characterize the binding of ribonuclease A (RNase A) to cytidi ne 2'-monophosphate (2'-CMP) and cytidine 3'-monophosphate (3'-CMP). We rep ort the changes in volume, Delta V and adiabatic compressibility, Delta K-S , that accompany the association of 2'-CMP and 3'-CMP with RNase A over a w ide temperature range. In general, the magnitudes of Delta V and Delta K-S are small. This results from compensation effects between the intrinsic and hydration terms. Specifically, a significant increase in the interaction v olume, V-I, compensates decreases in the intrinsic volume, V-M, and the the rmal volume, V-T, while an increase in the hydration term, Delta K-h, compe nsates a decrease in the intrinsic compressibility, K-M The volume and comp ressibility results suggest that 210 +/- 30 water molecules are released to the bulk state upon the binding of 2'-CMP or 3'-CMP to RNase A. Presumably , these water molecules originate from beyond the first coordination layer of the protein and the ligands. The binding of these ligands leads to a sim ilar to 15% decrease in the configurational entropy of the protein, suggest ing a decrease in the conformational dynamics of the protein upon ligand bi nding. The putative decrease in protein dynamics is consistent with the mea sured decrease in the intrinsic volume, V-M (1.4-2.1%), and the intrinsic c ompressibility, KM (5%), of RNase A upon the binding to 2'-CMP or 3'-CMP. T hus, RNase A becomes "smaller", "tighter", and "less mobile" upon binding e ither ligand. We discuss the relationship between macroscopic and microscop ic properties, in particular, how measured changes in volume, Delta V, and compressibility, Delta K-S, can be interpreted in terms of hydration proper ties of protein systems in their ligand-free and ligand-bound states.