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.
Highlights
Colicin E1 forms a voltage-dependent channel in the cytoplasmic membrane of target E. coli cells
Using sitedirected fluorescence labeling combined with our novel helical periodicity analysis method, we found that H6 and H7 are two separate and distinct amphipathic ␣-helices in the membraneassociated, pre-channel state and that H6 becomes overwound as a 310 helix upon membrane binding
The colicin E1 structure consists of 10 ␣-helices, where H8 and H9 are the hydrophobic helices that form the core of the soluble protein (Fig. 1A)
Summary
Colicin E1 forms a voltage-dependent channel in the cytoplasmic membrane of target E. coli cells. 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 ␣-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. 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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