title: Outer membrane protein F stabilised with minimal amphipol forms linear arrays and LPS-dependent 2D crystals.
abstract: Amphipols (APol) are polymers which can solubilise and stabilise membrane proteins (MP) in aqueous solutions. In contrast to conventional detergents, APol are able to keep MP soluble even when the free APol concentration is very low. Outer membrane protein F (OmpF) is the most abundant MP commonly found in the outer membrane (OM) of Escherichia coli. It plays a vital role in the transport of hydrophilic nutrients, as well as antibiotics, across the OM. In the present study, APol was used to solubilise OmpF to characterize its interactions with molecules such as lipopolysaccharides (LPS) or colicins. OmpF was reconstituted into APol by the removal of detergents using Bio-Beads followed by size-exclusion chromatography (SEC) to remove excess APol. OmpF/APol complexes were then analysed by SEC, dynamic light scattering (DLS) and transmission electron microscopy (TEM). TEM showed that in the absence of free APol-OmpF associated as long filaments with a thickness of ~6 nm. This indicates that the OmpF trimers lie on their sides on the carbon EM grid and that they also favour side by side association. The formation of filaments requires APol and occurs very rapidly. Addition of LPS to OmpF/APol complexes impeded filament formation and the trimers form 2D sheets which mimic the OM. Consequently, free APol is undoubtedly required to maintain the homogeneity of OmpF in solutions, but 'minimum APol' provides a new phase, which can allow weaker protein-protein and protein-lipid interactions characteristic of native membranes to take place and thus control 1D-2D crystallisation.
authors.affiliation: Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK.
authors.forename: Wanatchaporn
authors.initials: W
authors.lastname: Arunmanee
authors.forename: J Robin
authors.initials: JR
authors.lastname: Harris
authors.forename: Jeremy H
authors.initials: JH
authors.lastname: Lakey
mesh_terms: D003460:Crystallization; D008070:Lipopolysaccharides / Q000737:chemistry*; D046912:Multiprotein Complexes / Q000737:chemistry / Q000648:ultrastructure; D011108:Polymers / Q000737:chemistry*; D018272:Porins / Q000737:chemistry* / Q000648:ultrastructure*; D011437:Propylamines / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation; D013501:Surface-Active Agents / Q000737:chemistry*
chemical_list: D008070:Lipopolysaccharides; D046912:Multiprotein Complexes; C109237:OmpF protein; D011108:Polymers; D018272:Porins; D011437:Propylamines; D013501:Surface-Active Agents; C499017:amphipol A8-35