Biophysical Society Thematic Meeting | Ascona, Switzerland

Liposomes, Exosomes, and Virosomes: From Modeling Complex Membrane Processes to Medical Diagnostics and Drug Delivery

Poster Abstracts

26-POS Board 13 Investigating the Insertion and Folding of Membrane Proteins Into Lipid Bilayers Using a

Cell Free Expression System Nicola J. Harris .Paula J. Booth. King's College London, London, United Kingdom.

Membrane proteins play a vital role in many biological processes, and yet due to their instability in vitro remain poorly understood. This project aims to investigate the insertion and folding of membrane 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 E.coli proteins as they can offer important insights into other proteins, and thus facilitate the study of more biologically relevant targets. We have found that the E.coli rhomboid protease GlpG, and the Major Facilitator Superfamily (MFS) transport proteins LacY, XylE, GalP and GlpT, spontaneously insert into liposomes without the aid of an insertase such as SecYEG. This indicates that the innate hydrophobicity of these membrane proteins is sufficient for insertion into a bilayer. Spontaneously inserted GlpG is functional, as measured by its ability to cleave BODIPY-labeled casein, indicating that it is correctly folded. We have found that spontaneous insertion is highly influenced by the lipid composition of the liposomes. All the proteins tested to date prefer at least 50 mol% DOPG, and high DOPC has been found to be unfavourable for spontaneous insertion. On-going and future work will utilise rare codons to alter the rate of translation, and assess the effect this has on the final folded structure of membrane proteins. We will also examine whether the two helical domains of MFS transporters fold independently or cooperatively when expressed as two separate polypeptides. Preliminary results indicate that they fold independently. This work aids understanding into the folding and stability of membrane proteins.

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