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Orientation of the OmpF Porin in Planar Lipid Bilayers
Author(s) -
Ionescu Sandra A.,
Lee Sejeong,
Housden Nicholas G.,
Kaminska Renata,
Kleanthous Colin,
Bayley Hagan
Publication year - 2017
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201600644
Subject(s) - porin , periplasmic space , bacterial outer membrane , colicin , biophysics , membrane , lipid bilayer , extracellular , chemistry , protein structure , biochemistry , biology , escherichia coli , gene
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.