Investigating the Insertion and Folding of Membrane Transporters into Lipid Bilayers using a Cell Free Expression System

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Membrane proteins play a vital role in many biological processes, and yet due to their instability in vitro remain poorly understood. The Major Facilitator Superfamily (MFS) is a large group of transport proteins responsible for transporting a wide range of substrates. This project aims to investigate the insertion and folding of MFS proteins into lipid bilayers, using a commercial cell free expression system (PURExpress) in combination with synthetic liposomes of defined lipid composition. These studies will aid understanding of cooperative folding, folding intermediates, and the effects of the lipid bilayer on folding and insertion. We have chosen model prokaryotic transporters, as they can offer important insights into other proteins, and facilitate the study of more biologically relevant targets. We have found that the MFS transporters LacY, XylE, PepTso, GalP and GlpT spontaneously insert into liposomes, without the aid of an insertase such as SecYEG. This indicates that the innate hydrophobicity of these transporters is sufficient for insertion into a bilayer. Additionally, spontaneously inserted LacY is able to transport a fluorescent substrate across a Droplet Interface Bilayer, a good indication that it is folded correctly once inserted. This spontaneous insertion is highly influenced by the lipid composition of the liposomes. All the transporters tested to date prefer a lipid headgroup composition at least 50 % phosphatidylglycerol (PG), whereas bilayers containing phosphatidylcholine (PC) headgroups have the lowest spontaneous insertion yield. On-going work will investigate whether the two helical domains of MFS transporters fold independently or cooperatively when expressed as two separate polypeptides. Preliminary results indicate that they are able to fold independently.