Figure 4
Thermophoresis determined binding affinities of WT HtxB and D206A HtxB for phosphite and hypophosphite. Binding curves for (A) HtxB WT and (B) HtxB D206A with hypophosphite (black, pH 7.4; red, pH 5) and phosphite (blue, pH 5). Proteins were labelled with NT-647-NHS dye (10 nM final concentration) and MST performed in 40 mM citrate-phosphate buffer, 250 mM NaCl, 0.05% Tween-20, pH 7.4 or pH 5, with proteins subject to 22 seconds of thermophoresis. As appropriate, labeled proteins were mixed with serial dilutions of hypophosphite or phosphite. Calculated dissociation constants, Kd, are listed in Table 2. Error bars indicate the standard deviation from the mean of three independent titrations.
