The ligand affinity of proteins measured by isothermal denaturation kinetics

Analytical Biochemistry
Dennis E EppsP K Tomich

Abstract

An isothermal denaturation kinetic method was developed for identifying potential ligands of proteins and measuring their affinity. The method is suitable for finding ligands specific toward proteins of unknown function and for large-scale drug screening. It consists of analyzing the kinetics of isothermal denaturation of the protein-with and without the presence of potential specific ligands-as measured by long-wavelength fluorescent dyes whose quantum yield increases when bound to hydrophobic regions exposed upon unfolding of the proteins. The experimental procedure was developed using thymidylate kinase and stromelysin as target proteins. The kinetics of thermal unfolding of both of these enzymes were consistent with a pathway of two consecutive first-order rate-limiting steps. Reflecting the stabilizing effect of protein/ligand complexes, the presence of specific ligands decreased the 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 binding isotherm, the analysis of which yielded an accurate equilibrium constant for ligand binding. The method was validated by comparing its results with those obtained under the same conditions b...Continue Reading

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Citations

Nov 10, 2011·Assay and Drug Development Technologies·Guillermo SenisterraMasoud Vedadi
Nov 1, 2012·PloS One·Tobias BrandtDmitry B Veprintsev
Nov 9, 2011·Journal of Microbiological Methods·Uri Pick, Tatyana Rachutin-Zalogin
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Jun 10, 2010·Analytical Chemistry·Graham M WestMichael C Fitzgerald

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