Purification and NMR studies of [methyl-13C]methionine-labeled truncated methionyl-tRNA synthetase.
A procedure for the rapid purification of a truncated form of the Escherichia coli methionyl-tRNA synthetase has been developed. With this procedure, final yields of approximately 3 mg of truncated methionyl-tRNA synthetase per gram of cells, carrying the plasmid encoding the gene for the truncated synthetase [Barker, D.G., Ebel, J.-P., Jakes, R., & Bruton, C.J. (1982) Eur. J. Biochem. 127, 449], can be obtained. The catalytic properties of the purified truncated synthetase were found to be identical with those of the native dimeric and trypsin-modified methionyl-tRNA synthetases. A rapid procedure for obtaining milligram quantities of the enzyme is necessary before the efficient incorporation of stable isotopes into the synthetase becomes practical for physical studies. With this procedure, truncated methionyl-tRNA synthetase labeled with [methyl-13C]methionine was purified from an Escherichia coli strain auxotrophic for methionine and containing the plasmid encoding the gene for the truncated methionyl-tRNA synthetase. Both carbon-13 and proton observe-heteronuclear detect NMR experiments were used to observe the 13C-enriched methyl resonances of the 17 methionine residues in the truncated synthetase. In the absence of ligands, 13 of the 17 methionine residues could be resolved by carbon-13 NMR. Titration of the synthetase, monitoring the chemical shifts of resonances B and M (Figure 3), with a number of amino acid ligands and ATP yielded dissociation constants consistent with those derived from binding and kinetic data, indicating active site binding of the ligands under the conditions of the NMR experiment.(ABSTRACT TRUNCATED AT 250 WORDS)
Rosevear
P R
PR
Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston 77225.
D000596:Amino Acids; D000604:Amino Acyl-tRNA Synthetases / Q000302:isolation & purification*; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology; D009682:Magnetic Resonance Spectroscopy; D008718:Methionine-tRNA Ligase / Q000302:isolation & purification*; D008970:Molecular Weight; D009711:Nucleotides; D011487:Protein Conformation
D000596:Amino Acids; D009711:Nucleotides; D000604:Amino Acyl-tRNA Synthetases; D008718:Methionine-tRNA Ligase