Uncoupled Steps of the Colicin a Pore Formation Demonstrated by Bisulfide Bond Engineering

Abstract

Four disulfide bonds were engineered into the pore-forming domain of colicin A to probe the conformational changes associated with its membrane insertion and channel formation. The soluble pore-forming domain consists of 10 α-helices with two outer layers (helices 1, 2, and 3-7, respectively) sandwiching a middle layer of three helices (8-10). Helices 8 and 9 form a hairpin which is completely buried and consists of hydrophobic and neutral residues only. This helical hairpin has been hypothesized to be the membrane anchor. Each double-cysteine mutant possessing an individual disulfide bond, cross-linking either helices 1 to 9 (H1/H9), 5 to 6 (H5/H6), 7 to 8 (H7/H8), or 9 to 10 (H9/H10), resp..