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J. Biol. Chem., Vol. 281, Issue 43, 32375-32384, October 27, 2006

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Toward Elucidating the Membrane Topology of Helix Two of the Colicin E1 Channel Domain^*

Dawn White, Abdiwahab A. Musse, Jie Wang, Erwin London, and A. Rod Merrill¹

From the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada and the Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, New York 11794-5215

The membrane-bound closed state of the colicin E1 channel domain was investigated by site-directed fluorescence labeling using a bimane fluorophore attached to each single cysteine residue within helix 2 of each mutant protein. The fluorescence properties of the bimane fluorophore were measured for the membrane-associated form of the closed channel and included fluorescence emission maximum, fluorescence anisotropy, apparent polarity, surface accessibility, and membrane bilayer penetration depth. The fluorescence data show that helix 2 is an amphipathic -helix that is situated parallel to the membrane surface, but it is less deeply embedded within the bilayer interfacial region than is helix 1 in the closed channel. A least squares fit of the various data sets to a harmonic wave function indicated that the periodicity and angular frequency for helix 2 in the membrane-bound state are typical for an amphipathic -helix (3.8 ± 0.1 residues per turn and 94 ± 4°, respectively) that is located at an interfacial region of a membrane bilayer. Dual quencher analysis also revealed that helix 2 is peripherally membrane associated, with one face of the helix dipping into the interfacial region of the lipid bilayer and the other face projecting outwardly into the aqueous solvent. Finally, our data show that helices 1 and 2 remain independent helices upon membrane association with a short connector link (Tyr³⁶³–Gly³⁶⁴) and that short amphipathic -helices participate in the formation of a lipid-dependent, toroidal pore for this colicin.

Received for publication, June 20, 2006

^* This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (to A.R.M.) and by National Institutes of Health Grant GM 31986 (to E.L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 519-824-4120 (ext. 53806); Fax: 519-837-1802; E-mail: rmerrill{at}uoguelph.ca.

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