Fig. 7: In vitro proteolysis of the mutagenized FlaC.
From: Role of DegQ in differential stability of flagellin subunits in Vibrio vulnificus
a, b Proteolysis of FlaCM65V/M157V/M159L/M380L by DegQ. Mutagenized FlaC (1 μM), in which four Met residues at the 65, 157, 159, and 380th positions were substituted with the amino acids at the corresponding positions in FlaB (as shown in Supplementary Fig. 5b), were incubated with various concentrations of rDegQ (0, 0.12, 0.3, 0.48, 0.6, 0.72, 0.9, 1.08, and 1.2 μM). As described in Fig. 5, the remaining FlaCM65V/M157V/M159L/M380L in reaction mixtures were resolved in SDS-PAGE (the gel image), and their relative amounts were plotted against the given concentrations of rDegQ with the calculated values of EC50 (a). The average values of EC50 for proteolysis of the FlaCM65V/M157V/M380L by rDegQ were obtained from three independent proteolysis assays. For comparison, those of the original rFlaC and rFlaB were included (b). The error bars on the bar graph represent standard deviation. The P-values for comparison with the original FlaC were indicated (Student’s t-test; **P < 0.005). c, d Determination of the stability of the original and mutagenized flagellins. Thermal denaturation profiles of rFlaB, rFlaC, and rFlaCM65V/M157V/M159L/M380L were monitored by measuring the intensity of a fluorescent dye, SYPRO Orange, which had been labeled to the recombinant proteins (1 μM each), as described63. The melting temperatures (Tm) of each recombinant polypeptide, at which the lowest value of −dF/dT pointed, were obtained, as described in “Methods” (c). The average values of Tm of each recombinant polypeptide were obtained from three independent experiments (d). The error bars on the bar chart represent standard deviation. The P-values for comparison with the original rFlaC were indicated (Student’s t-test; **P < 0.005).
