An ion-channel-containing model membrane: structural determination by magnetic contrast neutron reflectometry.

ABSTRACT: To many biophysical characterisation techniques, biological membranes appear as two-dimensional structures with details of their third dimension hidden within a 5 nm profile. Probing this structure requires methods able to discriminate multiple layers a few Ångströms thick. Given sufficient resolution, neutron methods can provide the required discrimination between different biochemical components, especially when selective deuteration is employed. We have used state-of-the-art neutron reflection methods, with resolution enhancement via magnetic contrast variation to study an oriented model membrane system. The model is based on the Escherichia coli outer membrane protein OmpF fixed to a gold surface via an engineered cysteine residue. Below the gold is buried a magnetic metal layer which, in a magnetic field, displays different scattering strengths to spin-up and spin-down neutrons. This provides two independent datasets from a single biological sample. Simultaneous fitting of the two datasets significantly refines the resulting model. A ?-mercaptoethanol (?ME) passivating surface, applied to the gold to prevent protein denaturation, is resolved for the first time as an 8.2 ± 0.6 Å thick layer, demonstrating the improved resolution and confirming that this layer remains after OmpF assembly. The thiolipid monolayer (35.3 ± 0.5 Å), assembled around the OmpF is determined and finally a fluid DMPC layer is added (total lipid thickness 58.7 ± 0.9 Å). The dimensions of trimeric OmpF in isolation (53.6 ± 2.5 Å), after assembly of lipid monolayer (57.5 ± 0.9 Å) and lipid bilayer (58.7 ± 0.9 Å), are precisely determined and show little variation.

SUBMITTER: Holt SA 

PROVIDER: S-EPMC3035324 | biostudies-literature | 2009

REPOSITORIES: biostudies-literature