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Outer Membrane Protein F Stabilised with Minimal Amphipol Forms Linear Arrays and LPS-Dependent 2D Crystals

Lookup NU author(s): Wanatchaport Arunmanee, Professor Robin Harris, Professor Jeremy Lakey

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This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).


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 similar to 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.


Publication metadata

Author(s): Arunmanee W, Harris JR, Lakey JH

Publication type: Article

Publication status: Published

Journal: Journal of Membrane Biology

Year: 2014

Volume: 247

Issue: 9-10

Pages: 949-956

Print publication date: 01/10/2014

Online publication date: 01/03/2014

Acceptance date: 11/02/2014

ISSN (print): 0022-2631

ISSN (electronic): 1432-1424

Publisher: Springer

URL: http://dx.doi.org/10.1007/s00232-014-9640-5

DOI: 10.1007/s00232-014-9640-5


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