Fig. 3: Characterization of rPfGS lacking the first and second peptide inserts.
From: Distinct evolution of type I glutamine synthetase in Plasmodium and its species-specific requirement
a Schematic representations showing deletions of first and second peptide inserts. b HPLC chromatograms of ΔI1rPfGS and ΔI2rPfGS enzyme assays. Specific activities (mean ± SD shown below represent four different protein preparations. Lack of enzyme activity in ΔI2rPfGS was also verified with MnCl2 and for a prolonged incubation of 6 h. c Effect of MSO and PPT on ΔI1rPfGS activity. Individual data points are shown with the respective light shaded colors. d Feedback inhibition of ΔI1rPfGS in the presence of MgCl2 and MnCl2 at 5 mM concentrations of amino acids and AMP. For c and d percentage of activities (mean ± SD) with respect to control (without inhibitor/feedback inhibitor) are shown. n = 3 different protein preparations. e Comparison of Plasmodia and bacterial GS I dodecamer channels. Mt, St, and Hp structures were retrieved from PDB. f Comparison of rPfGS and ΔI1rPfGS thermal stabilities. Percentage of inhibition of the activities (mean ± SD) with respect to unexposed controls are shown. n = 3 different protein preparations. Recombinant proteins were exposed to the respective temperatures for one hour before performing assays at 37 °C. g Chromatograms showing the dissociation of oligomers in rPfGS and ΔI1rPfGS. Protein preparations were exposed to 37 °C or 42 °C for 15 min and subjected immediately to size-exclusion chromatography. h, Western analysis of rPfGS (~65 kDa) and ΔI1rPfGS (~63 kDa) in 12.7 and 15.9 ml elution volume fractions. i Estimation of rPfGS molecular weights eluted at 12.7 and 15.9 ml based on the elution of standard proteins. For g–i n = 2 independent protein preparations. j Unique hydrogen bond interactions of PfGS and ΔI2rPfGS are shown for subunit A. Four subunits are represented in green (subunit A), pink (subunit B), yellow (subunit C) and cyan (subunit D). Interactions without the subunit background are shown below. k Superimposition of MD simulation structures of two adjacent subunits from upper and lower hexamers of PfGS (grey) and ΔI2rPfGS (red). (n.s - not significant, **P < 0.01, ***P < 0.001, unpaired t-test; two-sided). Source data are provided as a Source Data file.
