MshE binds c-di-GMP.

<p>A. Isothermal Calorimetry was performed on purified proteins to determine the binding affinity for c-di-GMP. Three independent runs were included in the analysis, one representative run is depicted. VpsT is included as a positive control for c-di-GMP binding. B. Fluorescence thermal shift was utilized to investigate domains of MshE responsible for binding c-di-GMP. Fluorescence melt curves are displayed in the presence of 2mM ATP or c-di-GMP. Three independent experiments were performed in triplicate, with one representative experiment shown. C. Midpoints of melt curves are displayed in the presence of 2mM nucleotides or a buffer control. Three independent experiments were performed in triplicate, Significance was determined with a Oneway ANOVA followed by Dunnett’s Multiple Comparison test comparing to Buffer control. (*** p≤0.001, all others not significant).