Measuring the strength of binding of low molecular weight ligands to a target protein is a significant challenge to fragment-based drug discovery that must be solved. Thermal shift assays are uniquely suited for this purpose, due to the thermodynamic effects of a ligand on protein thermal stability. We show here how to implement a thermal shift assay, describing the basic features and analysis of the protein unfolding data. We then describe in detail the effects of a ligand on the observed stability of the protein to produce a shift in stability. The anatomy of ligand-induced thermal shift data is discussed in detail. We describe the unique aspects of concentration-response curves, the effect of protein unfolding energetics, and the stoichiometry of the interaction. We outline a typical assay development strategy for optimizing dye type and concentration, protein concentration, and buffer conditions. Guidelines are presented to demonstrate the limits of detection for weak-binding ligands, as applied to sulfonamide-based inhibitors of carbonic anhydrase II and applied to nucleotide binding to the death-associated protein kinase 1 catalytic domain.
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