Structure and dynamics of the membrane-embedded domain of LmrA investigated by coupling polarized ATR-FTIR spectroscopy and H-1/H-2 exchange

Bacterial LmrA, an integral membrane protein of Lactococcus lactis, confers multidrug resistance by mediating active extrusion of a wide variety of st ructurally unrelated compounds. Similar to its eucaryotic homologue P-gp, t his protein is a member of the ATP-binding cassette (ABC) superfamily. Diff erent predictive models, based on hydropathy profiles, have been proposed t o describe the structure of the ABC transporters in general and of LmrA in particular. We used polarized attenuated total reflection infrared spectros copy, combined with limited proteolysis, to investigate the secondary struc ture and the orientation of the transmembrane segments of LmrA. We bring th e first experimental evidence that the membrane-embedded domain of LmrA is composed of transmembrane-oriented a-helices. Furthermore, a new approach w as developed in order to provide information about membrane domain dynamics . Monitoring the infrared linear dichroism spectra in the course of H-1/H-2 exchange allowed to focus the recording of exchange rates on the membrane- embedded region of the protein only. This approach revealed an unusual stru ctural dynamics, indicating high flexibility in this antibiotic binding and transport region.