Anton Le Brun's Abstract

Electron microscopy and atomic force microscopy can provide structural information on the surface of a membrane and spectroscopic techniques tell us about the structure and dynamics of integral membrane proteins. However none of these techniques relate to what is happening in the 5 to 6 nm thick layer under the surface of the membrane. Only reflection methods can provide information about the distribution of materials on the axis perpendicular to the membrane surface (the z-axis). Neutron scattering can discriminate between hydrogen and its isotope deuterium, making neutron reflection a powerful tool for dissecting how lipid, protein and solvent relate to one another along the z-axis and provides a method in which certain components can be highlighted or made invisible by choosing the correct isotopic contrast. However in this work a novel method of magnetic contrast is also used. Buried under the gold surface to which the membrane layer is immobilised is a layer of a magnetic alloy. The magnetic layer has different scattering length densities depending on whether the neutrons are polarised to a spin up or to a spin down state. This provides two complementary but independent data sets on exactly the same biological samples allowing for unambiguous model fitting. The first step in immobilising membranes to gold surfaces is to immobilise a layer of 2-mercaptoethanol to prevent protein denaturation. Simultaneous fitting of data from the two spin states has resulted in the layer of 2-mercaptoethanol to be resolved for the first time, demonstrating the high resolution achieved by using the magnetic contrasts. The membrane system used is based on the outer membrane of the Gram-negative bacterial cell envelope. The protein component was Escherichia coli outer membrane protein F (OmpF), which is a homotrimeric porin acting as a molecular sieve allowing small molecule nutrients into the cell and keeping out large unwanted toxins. OmpF was directly immobilised to gold surfaces via an engineered thiol group. The lower lipid leaflet was also directly immobilised to the gold via a thiol head group. The bilayer was completed by the addition of an upper leaflet of lipids. The protein and lipid layers were fully characterised using simultaneous model fitting and Mote Carlo resampling and the high resolution data was found to be consistent with previous X-ray crystallography and atomic force microscopy data.