Role of Water in Protein−Ligand Interactions: Volumetric Characterization of the Binding of 2‘-CMP and 3‘-CMP to Ribonuclease A

Abstract

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, ΔV, and adiabatic compressibility, ΔKS, that accompany the association of 2‘-CMP and 3‘-CMP with RNase A over a wide temperature range. In general, the magnitudes of ΔV and ΔKS are small. This results from compensation effects between the intrinsic and hydration terms. Specifically, a significant increase in the interaction volume, VI, compensates decreases in the intrinsic volume, VM, and the thermal volume, VT, while an increase in the hydration term, ΔKh, compensates a decrease in the intrinsic compressibility, KM. 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, VM (1.4−2.1%), and the intrinsic compressibility, KM (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, ΔV, and compressibility, ΔKS, can be interpreted in terms of hydration properties of protein systems in their ligand-free and ligand-bound states.