Preparation and activity characterization of a type IV ABC transporter efflux pump in peptidiscs

Bacterial efflux pumps are membrane transporters that expel toxic compounds, including antibiotics, from the cell, contributing significantly to multidrug resistance. Among the seven major efflux pump families, transporters from the ATP-binding cassette (ABC) family are primary active systems that use ATP hydrolysis to extrude xenobiotics. Structural studies of these transporters have been advanced by the use of lipid-based reconstitution systems that preserve membrane protein functionality. While nanodiscs have enabled the determination of high-resolution structures, their reconstitution often requires careful optimization. In contrast, peptidisc - a small amphipathic peptide derived from apolipoprotein A-I - may offer a simplified alternative for stabilizing membrane proteins without the need of exogenous lipids. In this chapter, we describe the reconstitution into peptidiscs of PatAB, a type IV ABC transporter from Streptococcus pneumoniae that mediates fluoroquinolone resistance. We explain how mass photometry and size-exclusion chromatography with multi-angle light scattering (SEC-MALS) can be used to evaluate the molecular mass of the transporter in detergent and in peptidisc environments. Additionally, we explain how to reconstitute PatAB into nanodiscs and proteoliposomes, and compared the basal ATPase activity of the transporter in various environments. We highlight the utility of the peptidisc method as a versatile and efficient approach for reconstituting ABC transporters, enabling functional and structural analysis of drug resistance mechanisms.