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Liposomes, Exosomes, and Virosomes: From Modeling Complex
Membrane Processes to Medical Diagnostics and Drug Delivery
Poster Abstracts
82
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.