Visudtiphole Virak, Thomas Matthew B, Chalton David A, Lakey Jeremy H
Institute for Cell and Molecular Biosciences, University of Newcastle upon Tyne, Newcastle NE1 7RU, UK.
Biochem J. 2005 Dec 1;392(Pt 2):375-81. doi: 10.1042/BJ20051257.
The Escherichia coli OmpF (outer-membrane protein F; matrix porin) is a homotrimeric beta-barrel and a member of the bacterial porin superfamily. It is the best characterized porin protein, but has resisted attempts to refold it efficiently in vitro. In the present paper, we report the discovery of detergent-based folding conditions, including dodecylglucoside, which can create pure samples of trimeric OmpF. Whereas outer membrane LPS (lipopolysaccharide) is clearly required for in vivo folding, the artificially refolded and LPS-free trimer has properties identical with those of the outer-membrane-derived form. Thus LPS is not required either for in vitro folding or for structural integrity. Dimeric forms of OmpF have been observed in vivo and are proposed to be folding intermediates. In vitro, dimers occur transiently in refolding of trimeric OmpF and, in the presence of dodecylmaltoside, pure dimer can be prepared. This form has less beta-structure by CD and shows lower thermal stability than the trimer. Study of these proteins at the single-molecule level is possible because each OmpF subunit forms a distinct ion channel. Whereas each trimer contains three channels of equal conductance, each dimer always contains two distinct channel sizes. This provides clear evidence that the two otherwise identical monomers adopt different structures in the dimer and indicates that the asymmetric interaction, characteristic of C3 symmetry, is formed at the dimer stage. This asymmetric dimer may be generally relevant to the folding of oligomeric proteins with odd numbers of subunits such as aspartate transcarbamoylase.
大肠杆菌外膜蛋白F（OmpF；基质孔蛋白）是一种同三聚体β桶，属于细菌孔蛋白超家族。它是特征最明确的孔蛋白，但在体外有效重折叠的尝试中一直未能成功。在本文中，我们报告了基于去污剂的折叠条件的发现，包括十二烷基葡萄糖苷，其可以产生三聚体OmpF的纯样品。虽然体内折叠显然需要外膜脂多糖（LPS），但人工重折叠且无LPS的三聚体具有与外膜来源形式相同的性质。因此，LPS对于体外折叠或结构完整性都不是必需的。OmpF的二聚体形式已在体内观察到，并被认为是折叠中间体。在体外，三聚体OmpF重折叠过程中会短暂出现二聚体，并且在存在十二烷基麦芽糖苷的情况下，可以制备纯二聚体。通过圆二色光谱（CD）分析，这种形式的β结构较少，并且热稳定性低于三聚体。在单分子水平上研究这些蛋白质是可行的，因为每个OmpF亚基都形成一个独特的离子通道。每个三聚体包含三个电导率相等的通道，而每个二聚体始终包含两种不同的通道大小。这提供了明确的证据，表明两个原本相同的单体在二聚体中采用不同的结构，并表明在二聚体阶段形成了具有C3对称性特征的不对称相互作用。这种不对称二聚体可能普遍与具有奇数个亚基的寡聚蛋白（如天冬氨酸转氨甲酰酶）的折叠有关。
