Purification and ligand binding of EmrR, a regulator of a multidrug transporter - PubMed (original) (raw)

Purification and ligand binding of EmrR, a regulator of a multidrug transporter

A Brooun et al. J Bacteriol. 1999 Aug.

Abstract

EmrR, the repressor of the emrRAB operon of Escherichia coli, was purified to 95% homogeneity. EmrR was found to bind putative ligands of the EmrAB pump-2,4-dinitrophenol, carbonyl cyanide m-chlorophenylhydrazone, and carbonyl cyanide p-(trifluoro-methoxy)phenylhydrazone-with affinities in the micromolar range. Equilibrium dialysis experiments suggested one bound ligand per monomer of the dimeric EmrR.

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Figures

FIG. 1

FIG. 1

Affinity purification of EmrR. Protein was purified as described above. Samples were loaded onto a 12.5% acrylamide SDS-PAGE gel, run at 200 V until the dye front was at the bottom of the gel, and then stained with Coomassie brilliant blue R-250. Lanes 1 and 7, molecular mass markers; lane 2, crude lysate; lane 3, material passed through the IMAC column; lane 4, wash with 50 mM imidazole in binding buffer; lane 5, elution with 200 mM imidazole in binding buffer; lane 6, EmrR after cleavage with thrombin.

FIG. 2

FIG. 2

Equilibrium dialysis of EmrR bound to ligands. Ligand concentrations were obtained by measuring absorbance at 380 nm with a microtiter plate reader and referencing a corresponding calibration curve that produced a linear absorbance/concentration relationship in the 1 to 100 μM concentration range for each ligand. The data from equilibrium dialysis measurements were plotted to determine the K S (squares), _K_NS (diamonds), and _n_[_E_]t (x axis intercepts) for CCCP (A), FCCP (B), and DNP (C). The nonspecific data for DNP clustered around the origin and are not shown. Each data point is an average of three independent determinations. B, bound; F, free ligand.

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