FIG 10.

MBP-ToxT N106F has no change in binding affinity to P_tcpA after the addition of bicarbonate. MBP-ToxT WT and N106F binding to P_tcpA was analyzed using EMSA as previously described (37). Autoradiographs of EMSAs presented are representative of three or more independent experiments. Panel A is taken from our previous study of the effects of bicarbonate on ToxT binding (37). (Left) Binding reactions between MBP-ToxT WT (A) or MBP-ToxT N106F (B) and P_tcpA in lanes 1 to 7 took place in the absence of NaHCO₃. Reaction mixtures in lanes 8 to 14 were incubated in the presence of 36 mM NaHCO₃. Reaction mixtures in lanes 1 and 8 contained P_tcpA DNA in the absence of MBP-ToxT. Reaction mixtures in subsequent lanes contained titrations of MBP-ToxT with the concentrations indicated above the lanes. (Right) Binding curves for the autoradiographs at the left. Densitometry of autoradiographs was performed with ImageJ software. Circles represent percentages of P_tcpA bound by MBP-ToxT in the absence of bicarbonate, and the solid line corresponds to the binding curve for MBP-ToxT to P_tcpA, determined by the following equation using GraphPad Prism 5 software: % bound = B_max × [protein]^h/(K_d^h + [protein]^h), with the B_max constraint set to 100. The squares and dashed line represent percentages of P_tcpA bound by MBP-ToxT and the binding curve, respectively, in the presence of bicarbonate. The K_d for each condition is inset, and a significant difference between the best-fit values of each data set is denoted by an asterisk (P < 0.00025).