ORGANIC-SOLVENTS IDENTIFY SPECIFIC LIGAND-BINDING SITES ON PROTEIN SURFACES

Enzymes frequently recognize substrates and pharmaceutical drugs throu gh specific binding interactions in deep pockets on the protein surfac e. We show how the specificity-determining substrate binding site of h en egg-white lysozyme (HEWL) can be readily identified in aqueous solu tion by nuclear magnetic resonance spectroscopy using small organic so lvent molecules as detection probes. Exchange of magnetization between the H-1 nuclei of the protein and the ligands through dipole-dipole i nteractions is observed which allows the modeling of their position an d orientation at the binding site. Combined with site-specific binding constants measured by titration experiments with different organic so lvents, the method can provide important information for rational drug design. In addition, the lifetime of nonspecific interactions of HEWL with organic solvents is shown to be in the sub-nanosecond time range .