Atomic Detail Studies of P-Glycoprotein and Drug Permeation in Model Membranes

Abstract

The pathogenic Pseudomonas aeruginosa is a Gram-negative bacterium coexpressing two unique forms of lipopolysaccharides on its surface, the A and B bands. The A-band polysaccharides are thought to be translocated into the periplasm through an ATP-binding-cassette (ABC) transporter consisting of two subunits: a transmembranar protein, Wzm, and a nucleotide-binding protein, Wzt. Since Wzm and Wzt proteins possess no domains or motifs that are targeted for modifications by eukaryotic cellular machineries or exhibit any signal for organelles localization, we used Chinese hamster ovary (CHO) cells as a model system to study the protein complex structure and stoichiometry of Wzt protein in the absence or the presence of Wzm. We transfected the CHO cells with Wzt proteins fused to the green fluorescent protein (GFP2) or its variant yellow fluorescent protein (YFP) to study the protein complex structure of Wzt. A spectrally resolved two-photon microscope was used to obtain fluorescent images from transfected cells, and a novel spectral Fluorescence Resonance Energy Transfer (FRET) provided apparent FRET efficiency (Eapp) for each image pixel (Raicu et al., Nature Photonics, 2009). Distributions of FRET efficiencies were obtained from several cells, and the peak positions for the cells whose Eapp histograms showed single peak were binned to obtain a ''meta-histogram''. The meta-histograms were fitted to simulated distributions obtained from various theoretical models (Raicu et. al., Nature Photonics, 2009), to determine the stoichiometry and geometry of Wzt oligomers. Wzt formed rhombus-shaped homo-tertamers, which became square shaped upon co-expression of untagged Wzm.