PMID:23264635
| Citation |
Ho, D, Lugo, MR and Merrill, AR (2013) Harmonic analysis of the fluorescence response of bimane adducts of colicin E1 at helices 6, 7, and 10. J. Biol. Chem. 288:5136-48 |
|---|---|
| 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. |
| Links |
PubMed PMC3576118 Online version:10.1074/jbc.M112.436303 |
| Keywords |
Amino Acid Sequence; Anisotropy; Bacteriocins/chemistry; Colicins/chemistry; Cysteine/chemistry; Cytoplasm/metabolism; Disulfides; Escherichia coli/metabolism; Fluorescence Resonance Energy Transfer/methods; Microscopy, Fluorescence/methods; Molecular Conformation; Molecular Sequence Data; Mutagenesis; Protein Binding; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Temperature |
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