Purification and NMR studies of [13C-methyl]methionine-labeled truncated methionyl-tRNA synthetase

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Abstract
A procedure for the rapid purification of a truncated form of the Escherichia coli methionly-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., and 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 methionly-tRNA synthetases. A rapid procedure for obtaining milligram quantitities 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. The maximum chemical shift change of resonance B, in the presence of saturating concentrations of ligands, was found to be dependent on the exact nature of the amino acid side chain. Differences in the magnitude of the substrate-induced conformational change, detected by monitoring resonance B, amy be critical in triggering the recognition processes between cognate amino acid and synthetase. In contrast, the chemical shift of resonance M was found to be dependent on the negative charge introduced by the ligands at the aminoacyl adenylate site. This correlation is consistent with an induced-fit mechanism where portions of the binding site are formed as the various ligands are introduced into the aminoacyl adenylate site. Further studies on the solution structure of synthetase-ligand complexes will be useful in probing the structural mechanisms used in catalysis and amino acid discrimination.

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