Harmonic Analysis of the Fluorescence Response of Bimane Adducts of Colicin E1 at Helices 6, 7, and 10 |
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Authors: | Derek Ho Miguel R Lugo A Rod Merrill |
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Institution: | From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada |
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Abstract: | The pre-channel state of helices 6, 7, and 10 (Val447–Gly475 and Ile508–Ile522) 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. |
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Keywords: | Fluorescence Fluorescence Resonance Energy Transfer (FRET) Ion Channels Membrane Proteins Membrane Structure Cysteine-scanning Mutagenesis Helical Periodicity Analysis Bacteriocin Function Colicin Channel Structure Membrane Protein Topology |
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