Fluorescence polarization-based DNA binding assay.

Fluorescence polarization-based DNA binding experiments were performed with a Panvera Beacon 2000 fluorescence polarization system (Invitrogen) utilizing 5′-fluorescein-labeled DNA. Polarization (P) of such 5′-labeled DNA increases as a function of protein binding, and equilibrium dissociation constants are determined by curve fitting the values of millipolarization (P × 10⁻³) units against the protein concentration. To determine the effects of various ligands on MtrR-DNA binding, 1 nM 5′-fluoresceinated oligodeoxynucleotide duplexes containing the mtrC operator site in 1 ml binding buffer (20 mM Tris HCl [pH 8.0], 100 mM NaCl, and 2.5% glycerol) were titrated against increasing concentrations of purified MtrR in the presence or absence of the indicated concentrations of bile salt, and the resulting changes in polarization were measured. All the samples were excited at 490 nm, and their polarized emissions were measured at 530 nm. All the data were plotted using Kaleidagraph (Synergy Software) and Prism (GraphPad Software), and the resulting plots were fitted to the following equation: P = {(P_bound − P_free)[protein]/(K_D + [protein])} + P_free, where P is the polarization measured at a given protein concentration, P_free is the initial polarization of the free ligand, P_bound is the maximum polarization of specifically bound ligand, K_D is the equilibrium dissociation constant, and [protein] is the protein concentration. Nonlinear least-squares analysis was used to determine P_bound, and K_d. The reported binding constants are the average values from at least three independent experimental measurements.