Figure 5
Xanthine and urate are ligands for VvPecS. A-C. EMSA gels showing titrations of VvPecS (0.8 nM) and iSM DNA (0.9 fmol) with increasing concentration of ligands xanthine (A), urate (B), and guanosine (C). Free DNA (“D”) and protein-DNA complexes (“C”) identified at the right and ligand concentrations indicated above each lane. (D) Normalized percent complex formation as function of ligand concentration for urate and xanthine. Error bars represent standard deviation (SD) of three replicates. (E) Thermal stability of VvPecS. Fluorescence of SYPRO Orange bound to exposed hydrophobic regions of the protein as a function of temperature; fluorescence is normalized to the peak fluorescence. Unliganded VvPecS (black), and VvPecS incubated with urate (10 µM in yellow and 100 µM in orange), xanthine (10 µM in light green and 100 µM in dark green) and guanosine (100 µM in grey). The data are average relative fluorescence from triplicates. Uncropped versions of gels in panels A–C shown in Figs S10–S12.
