FIG 4.
Validation of cyclic di-GMP binding by microscale thermophoresis (MST). The PilZ effector Bd2717, the two-component response regulator Bd2402, and the acyl-CoA dehydrogenase Bd2924 were His tagged, produced, purified, and fluorescently labeled before subjected to MST analysis with increasing concentrations of cyclic di-GMP or cyclic di-AMP. The thermophoretic movement of the three proteins was affected by c-di-GMP but not by c-di-AMP. (A) Bd2717 bound c-di-GMP with Kd = 0.176 μM. (B) The thermophoretic movement of Bd2717 unaffected by c-di-AMP, provided as an example. (C) Bd2402 bound c-di-GMP with Kd = 0.399 μM. (C) Bd2924 bound c-di-GMP with Kd = 7.67 μM. Values are averages of three triplicates. Error bars indicate the standard deviations. MST plots of Bd2402 and Bd2924 with c-di-AMP are presented in Fig. S4 in the supplemental material.