Creating a double bilayer mimetic of the gram-negative bacterial envelope

The PqiABC complex has been implicated in phospholipid (PL) transport between the inner and outer membrane of gram-negative bacteria. This one complex spans the periplasm with components in both membranes, tethering them together. In a recent experiment, RB2220703, we proposed utilising this trans-envelope complex as a scaffold to create a planar double membrane architecture, providing a unique gram-negative envelope mimetic to probe envelope biology. Our Preliminary Neutron Reflectometry (NR) experiments confirmed re-constitution of the system but the distal bilayer remained liposomal and did not form a planar bilayar. However, in this configuration it allowed us to study PqiABC biology. We predicted PqiABC might be a proton motive force (PMF) driven PL pump based on structural homology. By altering the buffer pH, we were able to emulate the PMF across the liposomal membrane leading to a time dependent drop in the Scattering Length Density of the bilayer attached to the solid support. Suggestive of influx of hydrogenous material into the membrane, consistent with PMF driven PL transport. This proposal focuses on assessing conditions for rupturing the PqiAB proteoliposome to create a true double bilayer on the planar surface. This double bilayer configuration is required to properly assess changes within both membranes, a goal required to achieve our long term aim of using this double bilayer scaffold to study other envelope systems.