Outer Membrane Protein F Stabilised with Minimal Amphipol Forms Linear Arrays and LPS-Dependent 2D Crystals
Outer Membrane Protein F Stabilised with Minimal Amphipol Forms Linear Arrays and LPS-Dependent 2D Crystals
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DOI：
10.1007/s00232-014-9640-5
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发表时间：
2014-10-01
影响因子：
2.4
通讯作者：
Lakey, Jeremy H.
中科院分区：
文献类型：
--
作者：
Arunmanee, Wanatchaporn;Harris, J. Robin;Lakey, Jeremy H.
Amphipol（APol）是一种能够在水溶液中溶解和稳定膜蛋白（MP）的聚合物。与常规洗涤剂相比，即使当游离APol浓度非常低时，APol也能够保持MP可溶。外膜蛋白F（Outer membrane protein F，OmpF）是大肠杆菌（Escherichia coli，Escherichia coli）外膜中含量最丰富的蛋白质。它在跨OM运输亲水性营养素以及抗生素方面起着至关重要的作用。在本研究中，APol用于溶解OmpF以表征其与分子如脂多糖（LPS）或大肠杆菌素的相互作用。通过使用Bio-Beads去除去污剂，然后通过尺寸排阻色谱法（SEC）去除过量的APol，将OmpF重构为APol。然后通过SEC、动态光散射（DLS）和透射电子显微镜（TEM）分析OmpF/APol复合物。TEM显示，在不存在游离APol-OmpF的情况下，缔合为具有类似于6 nm的厚度的长丝。这表明OmpF三聚体位于碳EM网格上的侧面，并且它们也有利于并排缔合。细丝的形成需要APol，并且发生得非常迅速。向OmpF/APol复合物中加入LPS阻碍了细丝的形成，并且三聚体形成模拟OM的2D片层。因此，无疑需要游离APol来维持溶液中OmpF的均匀性，但“最小APol”提供了一种新相，其可以允许天然膜的特征性较弱的蛋白质-蛋白质和蛋白质-脂质相互作用发生，从而控制1D-2D结晶。
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.