title: Orientation of the OmpF Porin in Planar Lipid Bilayers.
abstract: 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.
authors.affiliation: Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford, OX1 3TA, UK.
authors.forename: Sandra A
authors.initials: SA
authors.lastname: Ionescu
authors.affiliation: Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford, OX1 3TA, UK.
authors.forename: Sejeong
authors.initials: S
authors.lastname: Lee
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.
authors.forename: Nicholas G
authors.initials: NG
authors.lastname: Housden
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.
authors.forename: Renata
authors.initials: R
authors.lastname: Kaminska
authors.affiliation: Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.
authors.forename: Colin
authors.initials: C
authors.lastname: Kleanthous
authors.affiliation: Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford, OX1 3TA, UK.
authors.forename: Hagan
authors.initials: H
authors.lastname: Bayley
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D000077422:Enrofloxacin; D029968:Escherichia coli Proteins / Q000737:chemistry*; D024841:Fluoroquinolones / Q000737:chemistry / Q000378:metabolism; D008051:Lipid Bilayers / Q000737:chemistry*; D008958:Models, Molecular; D018272:Porins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding
chemical_list: D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D024841:Fluoroquinolones; D008051:Lipid Bilayers; C109237:OmpF protein; D018272:Porins; D000077422:Enrofloxacin
keywords: antibiotics; colicin; membranes; planar lipid bilayer; porin