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Figure 1
Biochemical characterization of recombinant MtMetRS. (a) Size-exclusion chromatography of purified recombinant MtMetRS demonstrating that the enzyme is monomeric in solution. The Superdex 200 300/10 GL column was pre-calibrated with the protein standards thyroglobulin (670 kDa), γ-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa) and vitamin B12 (1.35 kDa). Upper insert, SDS–PAGE analysis of the eluates. (b) ATP–PPi exchange assay showing the catalytic activity of the recombinantly produced MtMetRS. (c) The velocity of ATP–PPi exchange is plotted as a function of methionine concentration. The data were fitted to the Michaelis–Menten equation to calculate Vmax and Km for methionine. (d) The velocity of ATP–PPi exchange is plotted as a function of ATP concentration. The data were fitted to the Michaelis–Menten equation to calculate Vmax and Km for ATP. (e) Thermal shift analysis of protein–ligand interaction. The Tm of the F-state MtMetRS was 55°C. The histogram displays the melting-temperature (ΔTm) shifts of MtMetRS upon incubation with various ligands: Met, ATP, AMP, ADO (adenosine), Met+AMP, Met+ADO and Met-ATP. The thermal shifts of two catalytically inactive mutants, E130A and K54A, were also measured. The concentration of the ligands was 200 µM and the concentration of MtMetRS was 2 µM. In the presence of SYPRO Orange, fluorescence (in relative fluorescence units; RFU) was recorded during heating from 10 to 85°C at a rate of 0.5°C every 30 s. The upper insert indicates the protein melting temperature (Tm).

IUCrJ
Volume 5| Part 4| July 2018| Pages 478-490
ISSN: 2052-2525
https://doi.org/10.1107/S2052252518008217