Determination of Real-Time Efflux Phenotypes in Escherichia coli AcrB Binding Pocket Phenylalanine Mutants Using a 1,2′-Dinaphthylamine Efflux Assay

To evaluate the importance of phenylalanine residues for substrate transport in the Escherichia coli efflux pump protein AcrB, we subjected Phe-to-Ala binding pocket mutants to a real-time efflux assay with the novel near-infrared lipophilic membrane probe 1,2′-dinaphthylamine (1,2′-DNA). All mutations, with the exception of F617A, led to considerable retardation of efflux. F610A was the point mutation with the most pronounced impact, followed by F628A, F615A, F136A, and F178A. This is the first study to demonstrate the importance of single phenylalanine residues within the AcrB binding pocket for real-time substrate transport

Increased quenching of 1,2′-DNA fluorescence at higher dye loading concentrations (DLCs) in strain 3-AG100.

<p>Depicted are relative fluorescence units (RFU) of deenergized cells preloaded with 1,2′-DNA after resuspension in fresh PPB. All experiments were done in triplicate. The error bars show the standard deviation.</p

Representative 1,2′-DNA efflux curves of 3-AG100-derived AcrB binding pocket mutants.

<p>After preloading with 4 µM 1,2′-DNA the cells were energized at 100 s with 50 mM glucose. Fluorescence intensity is given as relative fluorescence units (RFU) with preenergization levels adjusted to 100 RFU.</p