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 absorbance and CD spectroscopy in conjunction with high-pressure UV melting to detect and characterize the binding of ribonuclease A (RNase A) to cytidine 2‘-monophosphate (2‘-CMP) and cytidine 3‘-monophosphate (3‘-CMP). We report the changes in volume, Δ V, and adiabatic compressibility, Δ K S, that accompany the association of 2‘-CMP and 3‘-CMP with RNase A over a wide temperature range. In general, the magnitudes of Δ V and Δ K S are small. This results from compensation effects between the intrinsic and hydration terms. Specifically, a significant increase in the interaction volume, V I, compensates decreases in the intrinsic volume, V M, and the thermal volume, V T, while an increase in the hydration term, Δ K h, compensates a decrease in the intrinsic compressibility, K M . The volume and compressibility results suggest that 210 ± 40 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 ∼15% decrease in the configurational entropy of the protein, suggesting a decrease in the conformational dynamics of the protein upon ligand binding. The putative decrease in protein dynamics is consistent with the measured decrease in the intrinsic volume, V M (1.4−2.1%), and the intrinsic compressibility, K M (5%), of RNase A upon the binding to 2‘-CMP or 3‘-CMP. Thus, RNase A becomes “smaller”, “tighter”, and “less mobile” upon binding either ligand. We discuss the relationship between macroscopic and microscopic properties, in particular, how measured changes in volume, Δ V, and compressibility, Δ K S, can be interpreted in terms of hydration properties of protein systems in their ligand-free and ligand-bound states.