title: Membrane topology of the colicin E1 channel using genetically encoded fluorescence.
abstract: The membrane topology of the colicin E1 channel domain was studied by fluorescence resonance energy transfer (FRET). The FRET involved a genetically encoded fluorescent amino acid (coumarin) as the donor and a selectively labeled cysteine residue tethered with DABMI (4-(dimethylamino)phenylazophenyl-4'-maleimide) as the FRET acceptor. The fluorescent coumarin residue was incorporated into the protein via an orthogonal tRNA/aminoacyl-tRNA synthetase pair that allowed selective incorporation into any site within the colicin channel domain. Each variant harbored a stop (TAG) mutation for coumarin incorporation and a cysteine (TGT) mutation for DABMI attachment. Six interhelical distances within helices 1-6 were determined using FRET analysis for both the soluble and membrane-bound states. The FRET data showed large changes in the interhelical distances among helices 3-6 upon membrane association providing new insight into the membrane-bound structure of the channel domain. In general, the coumarin-DABMI FRET interhelical efficiencies decreased upon membrane binding, building upon the umbrella model for the colicin channel. A tentative model for the closed state of the channel domain was developed based on current and previously published FRET data. The model suggests circular arrangement of helices 1-7 in a clockwise direction from the extracellular side and membrane interfacial association of helices 1, 6, 7, and 10 around the central transmembrane hairpin formed by helices 8 and 9.
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada N1G 2W1.
authors.forename: Derek
authors.initials: D
authors.lastname: Ho
authors.forename: Miguel R
authors.initials: MR
authors.lastname: Lugo
authors.forename: Andrei L
authors.initials: AL
authors.lastname: Lomize
authors.forename: Irina D
authors.initials: ID
authors.lastname: Pogozheva
authors.forename: Suneel P
authors.initials: SP
authors.lastname: Singh
authors.forename: Adrian L
authors.initials: AL
authors.lastname: Schwan
authors.forename: A Rod
authors.initials: AR
authors.lastname: Merrill
mesh_terms: D000604:Amino Acyl-tRNA Synthetases; D003087:Colicins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003374:Coumarins / Q000737:chemistry; D003545:Cysteine / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000378:metabolism; D008051:Lipid Bilayers / Q000737:chemistry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017433:Protein Structure, Secondary; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism
chemical_list: D003087:Colicins; D003374:Coumarins; D005456:Fluorescent Dyes; D008051:Lipid Bilayers; C049748:4-dimethylaminophenylazophenyl-4'-maleimide; D004124:p-Dimethylaminoazobenzene; C030123:coumarin; D000604:Amino Acyl-tRNA Synthetases; D003545:Cysteine