Novel Bacterial Membrane Mimics: Investigating the Mechanosensitive Ion Channel MscL in the presence of antimicrobial molecules.

MscL is a highly conserved bacterial membrane protein that is part of the machinery that protects bacteria from osmotic shock. Gating of the ion channel is thought to be actuated by a force-through lipid mechanism, whereby a real strain in the membrane, caused by the influx of water in an osmotic shock, generates a membrane tension that is transmitted to the iris-like membrane protein.  Patch-clamp experiments have shown that MscL can be triggered to continuously gate by the presence of small amphiphillic molecules, such as lyso-PC. We are using neutron scattering and reflectivity measurements to investigate the mechanism of this gating when model bacterial membranes are challenged by lyso-PC or the amphipathic antimicrobial peptide Pexiganan.
Neutron scattering and reflectivity techniques are based on measuring interference effects in scattered neutrons with wavelengths of ~1-3 nm, which makes the techniques exquisitely sensitive to changes in thickness and water content (and hence neutron refractive index) of experimental models of bacterial membranes, which have thicknesses comparable to the neutron wavelength.
Reflectivity measurements require a planar model membrane of ~10 cm2 and we have developed a novel bilayer mimic, in which a POPC/POPG bilayer is suspended beneath a cationic surfactant monolayer at the air/water interface of a (Langmuir-like) trough. The suspended lipid bilayers are formed by the rupture of either liposomes or proteoliposomes beneath the cationic monolayer. Fits to our recent neutron reflectivity experiments show that a bilayer coverage of 99% can be achieved and that there is a greater than 5 nm thick water layer between the surfactant layer and the lipid bilayer. This water layer means that the suspended bilayer can fluctuate and that there is enough space to allow for membrane proteins to be inserted. To form the protein-containing bilayers we have prepared proteoliposomes containing mechanosensitive ion channels of large conductance, MscL, which we express through cell free protein expression directly into POPC/POPG liposomes.
In addition to these suspended bilayer mimics, we have also developed an experimental system in which the POPC/POPG/MscL bilayer is tethered to a gold layer, which sits on top of a thin permalloy film coated onto a silicon block. The silicon block acts as neutron window, and the permalloy layer means that we can exploit the two spin-states of the neutron to measure polarised reflectivity from the tethered bilayer system.
When these bilayer models are challenged with amphiphilic molecules that are known to exhibit antimicrobial properties (lyso-PC and pexiganan) we observe changes in the measured reflectivity and we are currently in the process of applying Bayesian inference to see if we can  extract the corresponding structural changes in the lipid/protein bilayer with sufficient reliability to understand the gating mechanism, and hence the antimicrobial activity of these molecules.