title: 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.
authors: Department of Biochemistry, University of Vermont, Burlington, VT 05405, USA. Electronic address: Jamie.Abbott@uvm.edu.
authors: Jamie A
authors: JA
authors: Abbott
authors: Department of Biochemistry, University of Vermont, Burlington, VT 05405, USA.
authors: Nathan M
authors: NM
authors: Livingston
authors: Department of Biochemistry, University of Vermont, Burlington, VT 05405, USA.
authors: Shawn B
authors: SB
authors: Egri
authors: Chemistry & Biochemistry Department, Norwich University, Northfield, VT 05663, USA.
authors: Ethan
authors: E
authors: Guth
authors: Department of Biochemistry, University of Vermont, Burlington, VT 05405, USA. Electronic address: Christopher.Francklyn@uvm.edu.
authors: Christopher S
authors: CS
authors: Francklyn
mesh_terms: D000412:Alanine-tRNA Ligase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D001578:Benzopyrans / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D005470:Fluorometry / Q000379:methods; D006643:Histidine-tRNA Ligase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009113:Muramidase / Q000737:chemistry / Q000378:metabolism; D044367:Phase Transition; D011485:Protein Binding; D058767:Protein Unfolding; D012345:RNA, Transfer, Amino Acid-Specific / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity; D013914:Threonine-tRNA Ligase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D046249:Transfer RNA Aminoacylation
chemical_list: D000596:Amino Acids; D001578:Benzopyrans; D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D005456:Fluorescent Dyes; D012345:RNA, Transfer, Amino Acid-Specific; D009113:Muramidase; D006643:Histidine-tRNA Ligase; D013914:Threonine-tRNA Ligase; D000412:Alanine-tRNA Ligase
keywords: Amino acid; Aminoacyl-tRNA synthetase; Differential scanning fluorimetry; Histidyl-tRNA synthetase; SYPRO Orange; tRNA