Single-molecule fluorescence studies on the conformational change of the ABC transporter MsbA

ATP-binding cassette (ABC) transporters are found in all forms of life from microbes to humans, and transport a wide variety of substrates across the cell membrane using the energy released from ATP hydrolysis and an alternating-access mechanism. MsbA is a homodimeric ABC exporter from Gramnegative bacteria, and transports amphipathic substrates including precursors of lipopolysaccharides from the inner leaflet to the outer leaflet of the cytoplasmic membrane. Despite extensive structural and functional studies, controversies remain regarding the dynamic properties of the conformational changes of MsbA during its transport cycle in the lipid environment. Here, we used single-molecule fluorescence resonance energy transfer (smFRET) to explore the dynamic behaviors of MsbA in detergent micelles, nanodiscs, and proteoliposomes. MsbA reconstituted into liposomes showed higher transition frequency between different states on the cytoplasmic side, whereas detergent-solubilized MsbA showed higher transition frequency on the periplasmic side. Three major states were identified from this smFRET study in the functional cycle of MsbA, including an intermediate conformation between the fully opened and fully closed cytoplasmic conformations, associated with both ATP binding and hydrolysis.