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Outer membrane protein F stabilised with minimal amphipol forms linear arrays and LPS-dependent 2D crystals.

Arunmanee W, Harris JR, Lakey JH - J. Membr. Biol. (2014)

Bottom Line: TEM showed that in the absence of free APol-OmpF associated as long filaments with a thickness of ~6 nm.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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK.

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.

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Effect of lipopolysaccharides on the structure of OmpF/APol complexes. a SEC analysis of size and homogeneity of OmpF/APol complexes in various conditions. (Solid line) LPS-free OmpF, (dashed line) OmpF+LPS, (dotted line) OmpF+LPS in 1 mM EDTA. In each case, the column was pre-equilibrated with detergent-free buffer. Profiles have been normalised to the same maximum. b SDS-PAGE analysis of LPS-free OmpF versus OmpF with added LPS showing characteristic ladder of OmpF bands resulting from LPS binding. The number of additional bands represents the increasing number of LPS attached to OmpF. EM study of OmpF/APol complexes in the presence and absence of LPS. c LPS-free OmpF/Apol. d OmpF+LPS/APol. e OmpF+LPS/APol/EDTA. Scale bars 100 nm
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Fig2: Effect of lipopolysaccharides on the structure of OmpF/APol complexes. a SEC analysis of size and homogeneity of OmpF/APol complexes in various conditions. (Solid line) LPS-free OmpF, (dashed line) OmpF+LPS, (dotted line) OmpF+LPS in 1 mM EDTA. In each case, the column was pre-equilibrated with detergent-free buffer. Profiles have been normalised to the same maximum. b SDS-PAGE analysis of LPS-free OmpF versus OmpF with added LPS showing characteristic ladder of OmpF bands resulting from LPS binding. The number of additional bands represents the increasing number of LPS attached to OmpF. EM study of OmpF/APol complexes in the presence and absence of LPS. c LPS-free OmpF/Apol. d OmpF+LPS/APol. e OmpF+LPS/APol/EDTA. Scale bars 100 nm

Mentions: Conventionally expressed, in vivo folded, OmpF is purified from the OM of E. coli which contains tightly-packed LPS in the outer leaflet. OmpF extracted from this membrane often co-purifies with the significant amounts of tightly bound LPS. This OmpF–LPS interaction is stable enough to be observed by SDS-PAGE analysis of native OmpF trimers and reveals a ladder pattern of LPS-bound OmpF bands (Fig. 2a) (Baboolal et al. 2008). The number of additional bands indicates the relative amount of LPS attached to the OmpF trimers. In the initial study, above, the samples had a low LPS content but were not conclusively LPS free. Since LPS molecules cross-link to adjacent LPS via divalent cations such as Ca2+ and Mg2+ (Schneck et al. 2010), the OmpF–LPS interaction may promote lateral OmpF association. In order to clearly investigate the effect of LPS on the formation of filaments, LPS-free OmpF was produced by refolding OmpF from denatured inclusion bodies. In the present study, LPS-free OmpF, OmpF+LPS and OmpF+LPS in EDTA were reconstituted into APol, and the complexes were fractionated by SEC. We used ‘rough’ Ra-LPS lacking the outer core and O-antigen to limit the interactions to the membrane region of OmpF and this was added to LPS-free OmpF at 1:5 OmpF/LPS molecular ratio prior to incubation with APol. The addition of 1 mM EDTA removed divalent cations from the buffer to inhibit LPS–LPS association. According to SEC elution profiles shown in Fig. 2b, LPS-free OmpF/APol complexes appeared to be monodisperse with a small amount of aggregate present, whereas the bulk of the OmpF+LPS/APol sample eluted as large aggregates at the void volume. Furthermore, when OmpF+LPS/APol complexes were formed in, and eluted in SEC column buffer containing EDTA, the amount of aggregates significantly decreased leaving a mixture of OmpF/APol particles with various sizes.Fig. 2


Outer membrane protein F stabilised with minimal amphipol forms linear arrays and LPS-dependent 2D crystals.

Arunmanee W, Harris JR, Lakey JH - J. Membr. Biol. (2014)

Effect of lipopolysaccharides on the structure of OmpF/APol complexes. a SEC analysis of size and homogeneity of OmpF/APol complexes in various conditions. (Solid line) LPS-free OmpF, (dashed line) OmpF+LPS, (dotted line) OmpF+LPS in 1 mM EDTA. In each case, the column was pre-equilibrated with detergent-free buffer. Profiles have been normalised to the same maximum. b SDS-PAGE analysis of LPS-free OmpF versus OmpF with added LPS showing characteristic ladder of OmpF bands resulting from LPS binding. The number of additional bands represents the increasing number of LPS attached to OmpF. EM study of OmpF/APol complexes in the presence and absence of LPS. c LPS-free OmpF/Apol. d OmpF+LPS/APol. e OmpF+LPS/APol/EDTA. Scale bars 100 nm
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Fig2: Effect of lipopolysaccharides on the structure of OmpF/APol complexes. a SEC analysis of size and homogeneity of OmpF/APol complexes in various conditions. (Solid line) LPS-free OmpF, (dashed line) OmpF+LPS, (dotted line) OmpF+LPS in 1 mM EDTA. In each case, the column was pre-equilibrated with detergent-free buffer. Profiles have been normalised to the same maximum. b SDS-PAGE analysis of LPS-free OmpF versus OmpF with added LPS showing characteristic ladder of OmpF bands resulting from LPS binding. The number of additional bands represents the increasing number of LPS attached to OmpF. EM study of OmpF/APol complexes in the presence and absence of LPS. c LPS-free OmpF/Apol. d OmpF+LPS/APol. e OmpF+LPS/APol/EDTA. Scale bars 100 nm
Mentions: Conventionally expressed, in vivo folded, OmpF is purified from the OM of E. coli which contains tightly-packed LPS in the outer leaflet. OmpF extracted from this membrane often co-purifies with the significant amounts of tightly bound LPS. This OmpF–LPS interaction is stable enough to be observed by SDS-PAGE analysis of native OmpF trimers and reveals a ladder pattern of LPS-bound OmpF bands (Fig. 2a) (Baboolal et al. 2008). The number of additional bands indicates the relative amount of LPS attached to the OmpF trimers. In the initial study, above, the samples had a low LPS content but were not conclusively LPS free. Since LPS molecules cross-link to adjacent LPS via divalent cations such as Ca2+ and Mg2+ (Schneck et al. 2010), the OmpF–LPS interaction may promote lateral OmpF association. In order to clearly investigate the effect of LPS on the formation of filaments, LPS-free OmpF was produced by refolding OmpF from denatured inclusion bodies. In the present study, LPS-free OmpF, OmpF+LPS and OmpF+LPS in EDTA were reconstituted into APol, and the complexes were fractionated by SEC. We used ‘rough’ Ra-LPS lacking the outer core and O-antigen to limit the interactions to the membrane region of OmpF and this was added to LPS-free OmpF at 1:5 OmpF/LPS molecular ratio prior to incubation with APol. The addition of 1 mM EDTA removed divalent cations from the buffer to inhibit LPS–LPS association. According to SEC elution profiles shown in Fig. 2b, LPS-free OmpF/APol complexes appeared to be monodisperse with a small amount of aggregate present, whereas the bulk of the OmpF+LPS/APol sample eluted as large aggregates at the void volume. Furthermore, when OmpF+LPS/APol complexes were formed in, and eluted in SEC column buffer containing EDTA, the amount of aggregates significantly decreased leaving a mixture of OmpF/APol particles with various sizes.Fig. 2

Bottom Line: TEM showed that in the absence of free APol-OmpF associated as long filaments with a thickness of ~6 nm.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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK.

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.

Show MeSH
Related in: MedlinePlus