Characterization of aminoacyl-tRNA synthetase stability and substrate interaction by differential scanning fluorimetry

•AARS protein stability can be evaluated by differential scanning fluorimetry (DSF).•DSF can be used for determining the stability of AARS enzyme substitutions.•AARS amino acid and inhibitor binding studies can be conducted with DSF.•Thermal stability can change as a function of tRNA binding and be measured by DSF. Differential scanning fluorimetry (DSF) is a fluorescence-based assay to evaluate protein stability by determining protein melting temperatures. Here, we describe the application of DSF to investigate aminoacyl-tRNA synthetase (AARS) stability and interaction with ligands. Employing three bacterial AARS enzymes as model systems, methods are presented here for the use of DSF to measure the apparent temperatures at which AARSs undergo melting transitions, and the effect of AARS substrates and inhibitors. One important observation is that the extent of temperature stability realized by an AARS in response to a particular bound ligand cannot be predicted a priori. The DSF method thus serves as a rapid and highly quantitative approach to measure AARS stability, and the ability of ligands to influence the temperature at which unfolding transitions occur.