Unraveling complex small-molecule binding mechanisms by using simple NMR spectroscopy.

Heteronuclear NMR spectroscopy provides a unique way to obtain site-specific information about protein-ligand interactions. Usually, such studies rely on the availability of isotopically labeled proteins, thereby allowing both editing of the spectra and ligand signals to be filtered out. Herein, we report that the use of the methyl SOFAST correlation experiment enables the determination of site-specific equilibrium binding constants by using unlabeled proteins. By using the binding of L- and D-tryptophan to serum albumin as a test case, we determined very accurate dissociation constants for both the high- and low-affinity sites present at the protein surface. The values of site-specific dissociation constants were closer to those obtained by isothermal titration calorimetry than those obtained from ligand-observed methods, such as saturation transfer difference. The possibility of measuring ligand binding to serum albumin at physiological concentrations with unlabeled proteins may open up new perspectives in the field of drug discovery.