tRNAfMet-induced conformational transition at the intersubunit domain of fluorescent-labeled methionyl-tRNA synthetase.
Conformational transition in methionyl-tRNA synthetase upon binding of tRNAfMet, whose binding shows strong negative cooporativity, was analyzed by fluorescence spectroscopy. The fluorescent probe N-[[(iodoacetyl)amino]ethyl]-5-naphthylamine-1-sulfonic acid (1,5-I-AEDANS) reacts with native methionyl-tRNA synthetase in a nearly stoichiometric amount (2 per dimer) without affecting enzyme activity. The probe is shown by controlled trypsinization to be located in a 130 amino acid fragment at the C-terminus joining the subunits. The emission and excitation spectra, rotational freedom, and solvent accessibility of the fluorophore in AEDANS-methionyl-tRNA synthetase are analyzed. The results suggest that the probe is localized in a nonpolar environment, nearly immobile relative to methionyl-tRNA synthetase yet fully accessible to the solvent. Upon binding of tRNAfMet, the fluorescence intensity in AEDANS-methionyl-tRNA synthetase was appreciably reduced without a shift in the emission or excitation spectra. Lifetime measurement shows that a static mechanism accounts for the observed quenching. Furthermore, the remaining emitting AEDANS becomes effectively shielded from solvent molecules. These results suggest an unsymmetric conformational transition at the intersubunit domains of the two subunits in methionyl-tRNA synthetase upon binding one molecule of tRNAfMet.
D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D001665:Binding Sites; D005456:Fluorescent Dyes; D007700:Kinetics; D046911:Macromolecular Substances; D008718:Methionine-tRNA Ligase / Q000378:metabolism*; D009282:Naphthalenesulfonates; D011485:Protein Binding; D011487:Protein Conformation; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012358:RNA, Transfer, Met; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D009282:Naphthalenesulfonates; D012346:RNA, Transfer, Amino Acyl; D012358:RNA, Transfer, Met; C024988:tRNA, formylmethionine-; C006421:1,5-I-AEDANS; D000604:Amino Acyl-tRNA Synthetases; D008718:Methionine-tRNA Ligase