Dissecting the Hydrophobic Effect on the Molecular Level: The Role of Water, Enthalpy, and Entropy in Ligand Binding to Thermolysin

Abstract

The hydrophobic effect is viewed as the driving force for the aggregation of nonpolar substances with extended lipophilic molecular surfaces in aqueous solution through the exclusion of water molecules from the formed interfaces. It is usually quoted to explain why an oil/water mixture spontaneously separates, why soluble proteins fold with a hydrophobic core and a hydrophilic outer surface, why membrane components assemble as lipid bilayers and micelles, why membrane proteins are accommodated in membrane segments, and why small molecules associate in protein binding pockets with mutual burial of hydrophobic surfaces. In the latter instance, it is a general strategy in medicinal chemistry to improve protein–ligand binding by increasing the ligand s hydrophobic surface which becomes buried in hydrophobic pockets of the target protein. In all cases, the hydrophobic effect is considered to be the major force of association. On the molecular level, this phenomenon is commonly attributed to the displacement of water molecules arranged around the hydrophobic surfaces, and entropic effects are made responsible to drive this association. The entropic profile is related to changes in the degree of ordering and the dynamic properties of the water molecules, which are assumed to be more disordered in the bulk water phase relative to where they were located prior to being displaced upon hydrophobic association. Recent studies have demonstrated, however, that hydrophobic interactions can originate either from enthalpyor entropy-driven binding, making simple explanations often presented for the hydrophobic effect insufficient. Also in computational design tools the handling of explicit water molecules has received increasing recognition. Tools such as WaterMap and Szmap try to take into account water structures in drug design and the properties of individual water molecules are discussed in terms of enthalpy and entropy. In order to obtain a better understanding of the hydrophobic effect on the molecular level and its role in protein– ligand binding, we embarked on a systematic study using thermolysin (TLN) as a model system. This thermostable bacterial zinc metalloprotease from Bacillus thermoproteolyticus exhibits three specificity pockets of predominantly hydrophobic nature (Scheme 1). It has been considered