The role of lipid bilayer composition in cell-free expressed ABC transporter folding

Cell-free protein expression systems provide a powerful tool to mechanistically probe how membrane composition, and the resulting biophysical properties, affect membrane protein (MP) complex folding and function. These systems can be designed without translocons, insertases, and chaperones commonly used to assist in membrane protein folding to isolate the impact of membrane properties on the folding process. In this study we investigate the ABC transporter, SapABCDF, in the gram-negative non-typeable Haemophilus influenzae (NTHI). This transporter sequesters heme and antimicrobial peptides (AMPs) from the periplasm into the cytoplasm via ATPase activity and understanding factors that contribute to MP complex folding and function is critical for developing antimicrobial treatments for NTHI and reduce the severity of infection. Using a cell-free protein expression system, we expressed each protein in the SapABCDF transporter in the presence of small unilamellar lipid vesicles (SUVs). We developed a sucrose flotation assay to evaluate protein colocalization to fluorescently labeled SUVs after cell-free expression and found that all components of the SapBCDF system express and colocalize to vesicles, suggesting their membrane integration and assembly. We then conducted a co-translational MP folding assay with a C-terminal mEGFP tag to determine membrane compositions that promote MP folding and expression. Analysis of cell-free expression products via western blot suggests that one transmembrane domain, SapC, expresses preferentially into SUVs containing phosphatidylethanolamine (PE). Ongoing studies aim to characterize transporter functionality and biophysically characterize the impact of bilayer biophysical properties on MP folding. Together, our results should improve our understanding of the biophysical requirements for properly folded and functioning ABC transporters.