Ionescu Sandra A, Lee Sejeong, Housden Nicholas G, Kaminska Renata, Kleanthous Colin, Bayley Hagan
Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford, OX1 3TA, UK.
Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.
Chembiochem. 2017 Mar 16;18(6):554-562. doi: 10.1002/cbic.201600644. Epub 2017 Feb 28.
The outer-membrane protein OmpF is an abundant trimeric general diffusion porin that plays a central role in the transport of antibiotics and colicins across the outer membrane of E. coli. Individual OmpF trimers in planar lipid bilayers (PLBs) show one of two current-voltage asymmetries, thus implying that insertion occurs with either the periplasmic or the extracellular end first. A method for establishing the orientation of OmpF in PLB was developed, based on targeted covalent modification with membrane-impermeant reagents of peripheral cysteine residues introduced near the periplasmic or the extracellular entrance. By correlating the results of the modification experiments with measurements of current asymmetry or the sidedness of binding of the antibiotic enrofloxacin, OmpF orientation could be quickly determined in subsequent experiments under a variety of conditions. Our work will allow the precise interpretation of past and future studies of antibiotic permeation and protein translocation through OmpF and related porins.
外膜蛋白OmpF是一种丰富的三聚体通用扩散孔蛋白，在抗生素和大肠杆菌素穿过大肠杆菌外膜的运输过程中起着核心作用。平面脂质双层（PLB）中的单个OmpF三聚体表现出两种电流-电压不对称性之一，因此意味着插入时要么是周质端先插入，要么是细胞外端先插入。基于用膜不透性试剂对在周质或细胞外入口附近引入的外周半胱氨酸残基进行靶向共价修饰，开发了一种确定OmpF在PLB中方向的方法。通过将修饰实验的结果与电流不对称性测量或抗生素恩诺沙星结合的侧别相关联，可以在各种条件下的后续实验中快速确定OmpF的方向。我们的工作将有助于准确解释过去和未来关于抗生素透过OmpF及相关孔蛋白的渗透和蛋白质转运的研究。
