The C-terminal half of the colicin A pore-forming domain is active in vivoand in vitro

The pore-forming domain of colicin A (pfColA) fused to a prokaryotic signal peptide (sp-pfColA) is transported across and inserts into the inner membr ane of Escherichia coli from the periplasmic side and forms a functional ch annel. The soluble structure of pfColA consists of a ten-helix bundle conta ining a hydrophobic helical hairpin. Here, we generated a series of mutants in which an increasing number of sp-pfColA alpha -helices was deleted. The se peptides were tested for their ability to form ion channels in vivo and in vitro. We found that the shortest sp-pfColA mutant protein that killed E scherichia coli was composed of the five last alpha -helices of sp-pfColA, whereas the shortest peptide that formed a channel in planar lipid bilayer membranes similar to that of intact pfColA was the protein composed of the last six alpha -helices. The peptide composed of the last five alpha -helic es of pfColA generated a voltage-independent conductance in planar lipid bi layer with properties very different from that of intact pfColA. Thus, heli ces 1 to 4 are unnecessary for channel formation, while helix 5, or some pa rt of it, is important but not absolutely necessary. Voltage-dependence of colicin is evidently controlled by the first four alpha -helices of pfColA. (C) 2001 Academic Press.