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. 2014 Jul 22;6(7):2162–2176. doi: 10.3390/toxins6072162

Figure 6.

Figure 6

The determination of binding affinities using microscale thermophoresis. Microscale thermophoresis measurements were done to quantify the binding of EGFP-fused TcdA1–1874 (♦), TcdA1–542 (■) and TcdA1102–1847 (○) to unlabeled CROPs. EGFP alone was used as the negative control (mock). The resulting thermophoresis signal was normalized and plotted against the respective CROP concentration. Data were fitted by the Hill slope, and the equilibrium binding constant KD was obtained. Data are presented as means ± SEM (n ≥ 4). The R-squared (R2) reflects the goodness of the respective fit. (A) The substrate Rac 1 was used as the positive control for binding to TcdA1–1874 (KD = 3.32 ± 0.36 μM); (B) Binding of TcdA1–1874 to TcdA CROPs resulted in a binding constant of 1.44 ± 0.07 μM (R2 = 0.98). The affinity of the shorter toxin fragments, TcdA1–542 and TcdA1102–1847, was less, with KD values of 2.96 ± 0.18 μM (R2 = 0.96) and 3.06 ± 0.18 μM (R2 = 0.89); (C) In contrast to the CROP domain of TcdA, the TcdB CROPs did not interact with the toxin fragments.