The specific incorporation of labelled aromatic amino acids into proteins through growth of bacteria in the presence of glyphosate

Abstract

Growth of Escherichia coli in the presence of glyphosate, an inhibitor of aromatic amino acid biosynthesis, has permitted the production of proton translocating ATPase that is specifically labelled with 5-fluorotryptophan. Five sets of tgF nuclear magnetic resonances are resolved. The use of glyphosphate should be of wide applicability in the preparation of proteins labelled in aromatic ammo acid residues for NMR studies.

EPR Spectroscopic Characterization of the Manganese Center and a Free Radical in the Oxalate Decarboxylase Reaction

“…Protein concentrations were measured by the method of Lowry (12). Incorporation of ring-deuterated tyrosine was achieved by growing cells in M9 minimal medium supplemented with 50 mg/liter phenylalanine, 35 mg/liter tryptophan, 5 mg/liter tyrosine, and 1 g/liter N-(phosphonomethyl)glycine (glyphosate) to inhibit de novo biosynthesis of aromatic amino acids (13). The culture was initiated with a limiting concentration of unlabeled tyrosine rather than the isotopically labeled form in order to minimize the possibility of deuterium incorporation into other amino acids through metabolic scrambling during biomass accumulation.…”

Observation of Asymmetry amongst Nucleotide Binding Sites of F1-ATPase of Escherichia coli by31P NMR Spectroscopy

“…F 1 -ATPase was prepared as described previously. [12][13][14][15][16] Enzyme was stored at -20 o C in column buffer, which contained Tris/HCl (50 mM, pH 7.4), 1 mM ATP, 1 mM DTT, 2 mM EDTA/Na and 10% glycerol. Activity was measured using a steady state coupled assay with pyruvate kinase and lactate dehydrogenase.…”

“…Many 19 F NMR studies of EF1 using fluorinated ligands, 12 fluoroaluminate complex, 13 fluoroberyllate complex 14 and internal fluorotryptophan labeling 15,16 characterized the nucleotide binding sites in the physiological condition. However, EF1 was not previously characterized with 31 P NMR.…”

Conformational Changes of Nucleotide Binding Sites Following Sequential Addition of ADP to Nucleotide-depleted F1-ATPase of Escherichia coli Investigated with31P NMR Spectroscopy

“…Many 19 F NMR studies of EF1 using fluorinated ligands, 12 fluoroaluminate complex, 13 fluoroberyllate complex 14 and internal fluoro-tryptophan labeling 15,16 were performed to characterize nucleotide binding sites of F 1 -ATPase of E. coli in the physiological condition.…”

“…As molecular weight of F 1 -ATPase (380 K) is very large compared to that of adenylate kinase (32 K), adenylate kinase-like activity of F 1 -ATPase cannot be from contaminated adenylate kinase after gel filtration in the preaparation. [13][14][15][16] The resonance at around 5 ppm must be originated from a transfer of phosphoryl group of ADP to a possible acceptor molecule in the medium, which is methanol added as a stabilizer of F 1 -ATPase. Comparison with the 31 P NMR spectrum of alkyl phosphate could confirm the presence of methyl phosphate.…”