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

Abstract

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.

Isothermal Analysis of ThermoFluor Data can readily provide Quantitative Binding Affinities

“…Because of the complexity associated with correctly replicating this analysis, it is often cited in modern studies but not frequently used: DSF is most popular as a qualitative test rather than a quantitative test, with the majority of literature reports reporting T m -shifts as shown in Fig. 1B but not attempting to extract binding constants- 8,11,28,29,41–44 . Collectively this has led to a general consensus that the observed T m shifts “cannot be readily transformed into binding affinities” 45 .…”

Allosteric activation of SARS-CoV-2 RdRp by remdesivir triphosphate and other phosphorylated nucleotides

“…To test whether Nsp12 R is misfolded, we used several approaches. First , we assessed the Nsp12 thermal stability using differential scanning fluorimetry ( Abbott et al, 2017 ). We recorded melting temperatures (T m ) of 41.3 °C for Nsp12 R and 47.3 °C for Nsp12 A ( Figure S3A ); T m of 43.6 °C was reported for another E. coli -expressed Nsp12 ( Peng et al, 2020 ).…”

“…To test whether Nsp12 R is misfolded, we used several approaches. First, we assessed the Nsp12 thermal stability using differential scanning fluorimetry (Abbott et al, 2017). We recorded melting temperatures (Tm) of 41.…”

Aminoacyl-tRNA synthetases

“…In the first set of contributions (First [16], Cvetesic [17], and Schwartz [18]) methods are described for continuous analysis of the aminoacylation and editing reactions (First)[16], in vitro /in vivo analysis of AARS fidelity and editing (Cvetesic [16], Schwartz [18]), and the interaction of AARSs with inhibitors (Saint-Leger)[19]. This is followed by new approaches for synthetase structure, including AARS complex formation analysis by SAXS (Cantara)[20] and NMR (Cho)[21]; stability analysis by thermal shift profiles (Abbott)[22]; phosphorylation status of AARSs (Fox)[23]; and X-ray crystallography (Fang)[24]. Next are featured contributions examining methods for localizing AARS functions in cells, including the various compartments of yeast (Debard)[25]; in the nucleus (Shi)[26], in the mitochondria (Carapito)[27], and in various domains of the bacterial cell (Zhao)[28].…”