title: Harmonic analysis of the fluorescence response of bimane adducts of colicin E1 at helices 6, 7, and 10.
abstract: The pre-channel state of helices 6, 7, and 10 (Val(447)-Gly(475) and Ile(508)-Ile(522)) of colicin E1 was investigated by a site-directed fluorescence labeling technique. A total of 44 cysteine variants were purified and covalently labeled with monobromobimane fluorescent probe. A variety of fluorescence properties of the bimane fluorophore were measured for both the soluble and membrane-bound states of the channel peptide, including the fluorescence emission maximum, fluorescence anisotropy, and membrane bilayer penetration depth. Using site-directed fluorescence labeling combined with our novel helical periodicity analysis method, the data revealed that helices 6, 7, and 10 are separate amphipathic α-helices with a calculated periodicity of T = 3.34 ± 0.08 for helix 6, T = 3.56 ± 0.03 for helix 7, and T = 2.99 ± 0.12 for helix 10 in the soluble state. In the membrane-bound state, the helical periodicity was determined to be T = 3.00 ± 0.15 for helix 6, T = 3.68 ± 0.03 for helix 7, and T = 3.47 ± 0.04 for helix 10. Dual fluorescence quencher analysis showed that both helices 6 and 7 adopt a tilted topology that correlates well with the analysis based on the fluorescence anisotropy profile. These data provide further support for the umbrella model of the colicin E1 channel domain.
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
authors.forename: Derek
authors.initials: D
authors.lastname: Ho
authors.forename: Miguel R
authors.initials: MR
authors.lastname: Lugo
authors.forename: A Rod
authors.initials: AR
authors.lastname: Merrill
mesh_terms: D000595:Amino Acid Sequence; D016880:Anisotropy; D001430:Bacteriocins / Q000737:chemistry; D003087:Colicins / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000378:metabolism; D004220:Disulfides; D004926:Escherichia coli / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013696:Temperature
chemical_list: D001430:Bacteriocins; D003087:Colicins; D004220:Disulfides; D003545:Cysteine