O'Keeffe A H, East J M, Lee A G
Division of Biochemistry and Molecular Biology, School of Biological Sciences, University of Southampton, Southampton SO16 7PX, United Kingdom.
Biophys J. 2000 Oct;79(4):2066-74. doi: 10.1016/S0006-3495(00)76454-X.
The outer membrane porin OmpF from Escherichia coli has been reconstituted into lipid bilayers of defined composition, and fluorescence spectroscopy is used to characterize its interaction with the surrounding lipid. OmpF is a trimer within the membrane. It contains two Trp residues per monomer, Trp(214) at the lipid-protein interface and Trp(61) at the trimer interface. The fluorescence of Trp-214 in the mutant W61F is quenched by dibromostearoylphosphatidylcholine (di(Br(2)C18:0)PC), whereas the fluorescence of Trp(61) in the mutant W214F is not quenched by di(Br(2)C18:0)PC when fluorescence is excited directly through the Trp rather than through the Tyr residues. Measurements of relative fluorescence quenching for OmpF reconstituted into mixtures of lipid X and di(Br(2)C18:0)PC have been analyzed to give the binding constant of lipid X for OmpF, relative to that for dioleoylphosphatidylcholine (di(C18:1)PC). The phosphatidylcholine showing the strongest binding to OmpF is dimyristoyloleoylphosphatidylcholine (di(C14:1)PC) with binding constants decreasing with either increasing or decreasing fatty acyl chain length. Comparison with various theories for hydrophobic matching between lipids and proteins suggests that in the chain length range from C14 to C20, hydrophobic matching is achieved largely by distortion of the lipid bilayer around the OmpF, whereas for chains longer than C20, distortion of both the lipid bilayer and of the protein is required to achieve hydrophobic matching. Phosphatidylcholine and phosphatidylethanolamine bind with equal affinity to OmpF, but the affinity for phosphatidylglycerol is about half that for phosphatidylcholine.
来自大肠杆菌的外膜孔蛋白OmpF已被重组到特定组成的脂质双层中，并且使用荧光光谱法来表征其与周围脂质的相互作用。OmpF在膜内是三聚体。每个单体包含两个色氨酸残基，位于脂质 - 蛋白质界面的Trp(214)和位于三聚体界面的Trp(61)。当直接通过色氨酸而不是通过酪氨酸残基激发荧光时，突变体W61F中Trp - 214的荧光被二溴硬脂酰磷脂酰胆碱（di(Br(2)C18:0)PC）淬灭，而突变体W214F中Trp(61)的荧光不被di(Br(2)C18:0)PC淬灭。对重组到脂质X和di(Br(2)C18:0)PC混合物中的OmpF的相对荧光淬灭测量进行了分析，以给出脂质X对OmpF的结合常数，相对于二油酰磷脂酰胆碱（di(C18:1)PC）的结合常数。与OmpF结合最强的磷脂酰胆碱是二肉豆蔻酰油酰磷脂酰胆碱（di(C14:1)PC），结合常数随着脂肪酰链长度的增加或减少而降低。与关于脂质和蛋白质之间疏水匹配的各种理论的比较表明，在C14至C20的链长范围内，疏水匹配主要通过OmpF周围脂质双层的扭曲来实现，而对于长于C20的链，需要脂质双层和蛋白质的扭曲来实现疏水匹配。磷脂酰胆碱和磷脂酰乙醇胺以相等的亲和力与OmpF结合，但对磷脂酰甘油的亲和力约为磷脂酰胆碱的一半。
