Thermal shift assay for protein–ligand dissociation constant determination

Accurate determination of protein-ligand affinity is essential for drug design when new compounds should be ranked according to their dissociation constants Kd. The thermal shift assay (TSA) is a technique used both for protein stability and ligand Kd measurements. Here, we review the principles of the TSA and describe the advantages and limitations of its use for protein-ligand affinity measurements. The TSA is a very powerful technique to determine protein-ligand affinity because it accurately measures for extremely large dynamic range of affinities from milimolar to picomolar in a single experiment. To help users analyze TSA data for protein-ligand affinity determination, we recently introduced a web-based application, Thermott, that provides solutions to thermodynamic equations that include the enthalpies and heat capacities of the protein unfolding and protein-ligand binding reactions.