The ligand affinity of proteins measured by isothermal denaturation kinetics

An isothermal denaturation kinetic method was developed for identifying pot ential ligands of proteins and measuring their affinity. The method is suit able for finding Ligands specific toward proteins of unknown function and f or large-scale drug screening. It consists of analyzing the kinetics of iso thermal denaturation of the protein-with and without the presence of potent ial specific ligands-as measured by long-wavelength fluorescent dyes whose quantum yield increases when bound to hydrophobic regions exposed upon unfo lding of the proteins. The experimental procedure was developed using thymi dylate kinase and stromelysin as target proteins, The kinetics of thermal u nfolding of both of these enzymes were consistent with a pathway of two con secutive first-order rate-limiting steps. Reflecting the stabilizing effect of protein/ligand complexes, the presence of specific ligands decreased th e value of the rate constants of both steps in a dose-dependent manner. The dependence of the rate constants on Ligand concentration obeyed a simple b inding isotherm, the analysis of which yielded an accurate equilibrium cons tant for Ligand binding. The method was validated by comparing its results with those obtained under the same conditions by steady-state fluorescence spectroscopy, circular dichroism, and uv spectrophotometry: The correspondi ng rate constants were comparable for each of the analytical detection meth ods. (C) 2001 Academic Press.