(176g) Co-Translational Membrane Protein Folding into Supported Hybrid Polymer-Lipid Membrane Maintains Protein Orientation and Mobility

Membrane proteins represent a priority target for fundamental biophysical studies and therapeutic development, but we know relatively little about them compared to soluble proteins. The complex lipid environment where they natively reside is a fluid membrane allowing for critical protein-lipid interactions that carry out important cellular functions. Studying them within the complete cellular environment makes it hard to isolate specific processes. Reconstitution of membrane proteins into synthetic vesicles allows for easy manipulation of membrane properties and enables new insight into membrane-membrane protein relationships. Researchers have begun using cell-free synthesis to express membrane proteins in the presence of liposomes to mimic the native co-translationally folding of a protein, and recently demonstrated the functional insertion of various membrane proteins into vesicles containing both phospholipids and di-block copolymers. To adapt this system to be compatible with many biophysical characterization tools and applications requiring planar geometry, we report here the development of a cell-free expression system for the co-translational insertion of membrane proteins into a supported, hybrid, lipid bilayer incorporating diblock copolymers. We use hybrid vesicles made with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 0 – 35 mol% poly(ethylene oxide)-b-poly(butadiene) to form a contiguous supported bilayer and analyze the effect of polymer concentration on lipid diffusion and membrane stiffness using fluorescence microscopy and quartz crystal microbalance with dissipation (QCM-D), respectively. Then, we use cell-free expression to synthesize a model membrane protein, the mechanosensitive channel of large conductance (MscL) containing a GFP folding reporter, directly into the supported bilayer. We found that these proteins could be translated into the hybrid bilayer and confirmed that that the proteins were folded correctly using the fluorescence reporter. Conveniently, we used the fluorescent reporter to assess the mobility of the protein in the planar bilayer using total internal reflection fluorescence (TIRF) microscopy and single particle tracking. We found that the presence of diblock copolymer in supported bilayers promotes protein mobility in comparison to lipid-only bilayers. We then used a protease cleavage assay to determine the protein orientation and found that it matches that of proteins directly translated into liposomes. By maintaining this orientation in the planar platform, we can then assess the functionality of the protein through its channel activity for future biophysical studies of protein-lipid interactions. Our platform allows for the high-throughput study of membrane proteins on a chip to elucidate biophysical behavior in a planar conformation.