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.

Gram-negative trimeric porins have specific LPS binding sites that are essential for porin biogenesis

“…In an AFM study, Schabert and Engel (40) showed that the addition of loosely bound LPS changed 2D crystals from rectangular to hexagonal, whereas EM (24) and AFM (39) studies of bacterial outer membranes revealed quasicrystalline arrangements of porins and LPS in vivo. Furthermore, OmpF stabilized by the polymeric detergent amphipol can be rapidly induced to form dense 2D arrays by the addition of excess LPS (35). These data, combined with the specific interactions revealed here, explain how porins and LPS form tight assemblies on the surfaces of gram-negative bacteria that preserve the necessary stability and impermeability of the OM.…”

“…S4). We have previously observed the ability of LPS to promote 2D crystallization of E. coli OmpF trimers, and the bridging positions of the site B (and possibly site C) LPS molecule help explain this behavior (35).…”

Amphipols for Each Season

“…Studies with tOmpA (Zoonens et al 2007), BR (Gohon et al 2008), the bc 1 complex (Charvolin et al 2014), E. coli ’s outer membrane protein F (OmpF) (Arunmanee et al 2014), and the ExbB–ExbD complex (Sverzhinsky et al 2014) have shown that the presence of some free APol particles is essential to keeping MP/APol complexes monodisperse, a 1:1 ratio between bound and free APol being typically sufficient (cf. Fig.…”

The Use of Amphipols for NMR Structural Characterization of 7-TM Proteins

“…by washing amphipol-solubilized BR with amphipol-free buffer while being immobilized on a Ni-NTA column, leads to formation of very large protein-amphipol particles (Fig. 1c) (also see (Arunmanee et al, 2014)). In agreement with previous findings these large particles can be converted into regular sized particles to some extent by the addition of free amphipols (Fig.…”