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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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Progression of filament formation by OmpF/APol complexes. a Size distribution of OmpF/APol complexes by intensity measured at 30 °C by DLS from 15 min to 6 days after SEC. b Structural models of possible OmpF filaments showing ‘top’ and ‘side’ views. Electron microscopy of OmpF/APol studied at different times after SEC. c After 10 min. Single particles of OmpF trimer (arrowheads) were present. d After 10 min. OmpF pores (arrowheads) can be seen on filaments. e After 1 day. f After 1 week. Scale bars 100 nm. The insets in c, d are at ×2 higher magnification
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Fig3: Progression of filament formation by OmpF/APol complexes. a Size distribution of OmpF/APol complexes by intensity measured at 30 °C by DLS from 15 min to 6 days after SEC. b Structural models of possible OmpF filaments showing ‘top’ and ‘side’ views. Electron microscopy of OmpF/APol studied at different times after SEC. c After 10 min. Single particles of OmpF trimer (arrowheads) were present. d After 10 min. OmpF pores (arrowheads) can be seen on filaments. e After 1 day. f After 1 week. Scale bars 100 nm. The insets in c, d are at ×2 higher magnification

Mentions: In the previous experiments, all complexes for TEM study were prepared the day before, and thus the rate of formation of OmpF/APol complexes was unknown. To measure the kinetics of OmpF/APol filament formation after the removal of free APol, the size distribution of LPS-free OmpF/APol complexes after SEC was monitored by DLS at 30 °C as a function of time. The size distributions were analysed by intensity as this mode is more sensitive to large particles in the samples. The size of OmpF trimer in conventional detergents measured by DLS is approximately 10 nm (data not shown). DLS size distribution data of OmpF/APol complexes suggested that the number of particles with sizes above 10 nm increased over time while the number of single OmpF trimers/APol decreased (Fig. 3a). Even though we measured freshly prepared complexes, a small amount of large particles was already present after 15 min. Therefore, it is evident that self-association of OmpF in the absence of free APols occurred very rapidly.Fig. 3


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)

Progression of filament formation by OmpF/APol complexes. a Size distribution of OmpF/APol complexes by intensity measured at 30 °C by DLS from 15 min to 6 days after SEC. b Structural models of possible OmpF filaments showing ‘top’ and ‘side’ views. Electron microscopy of OmpF/APol studied at different times after SEC. c After 10 min. Single particles of OmpF trimer (arrowheads) were present. d After 10 min. OmpF pores (arrowheads) can be seen on filaments. e After 1 day. f After 1 week. Scale bars 100 nm. The insets in c, d are at ×2 higher magnification
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4196048&req=5

Fig3: Progression of filament formation by OmpF/APol complexes. a Size distribution of OmpF/APol complexes by intensity measured at 30 °C by DLS from 15 min to 6 days after SEC. b Structural models of possible OmpF filaments showing ‘top’ and ‘side’ views. Electron microscopy of OmpF/APol studied at different times after SEC. c After 10 min. Single particles of OmpF trimer (arrowheads) were present. d After 10 min. OmpF pores (arrowheads) can be seen on filaments. e After 1 day. f After 1 week. Scale bars 100 nm. The insets in c, d are at ×2 higher magnification
Mentions: In the previous experiments, all complexes for TEM study were prepared the day before, and thus the rate of formation of OmpF/APol complexes was unknown. To measure the kinetics of OmpF/APol filament formation after the removal of free APol, the size distribution of LPS-free OmpF/APol complexes after SEC was monitored by DLS at 30 °C as a function of time. The size distributions were analysed by intensity as this mode is more sensitive to large particles in the samples. The size of OmpF trimer in conventional detergents measured by DLS is approximately 10 nm (data not shown). DLS size distribution data of OmpF/APol complexes suggested that the number of particles with sizes above 10 nm increased over time while the number of single OmpF trimers/APol decreased (Fig. 3a). Even though we measured freshly prepared complexes, a small amount of large particles was already present after 15 min. Therefore, it is evident that self-association of OmpF in the absence of free APols occurred very rapidly.Fig. 3

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