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 alpha-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), respectively, is unable to promote K+ efflux from sensitive Escherichia coli cells. Activity can be restored by addition of a reducing agent. In vitro studies with brominated lipid vesicles and planar lipid bilayers show that the disulfide bond which connects the helices 1 to 9 prevents colicin A membrane insertion, whereas the other disulfide bond mutants insert readily into lipid vesicles. All of the engineered bridges prevented the formation of a conducting channel in the presence of a membrane potential. This novel approach indicates that membrane insertion and channel formation are two separate steps. Moreover, the effects of the distance constraints introduced by the different disulfide bonds on colicin A activity indicate that the helical pair 1 and 2 moves away from the other helices upon membrane insertion. Helices 3-10 remain associated together. As a consequence, the results imply that the helical hairpin lies parallel to the membrane surface. In contrast, induction of the colicin channel by the membrane potential requires a profound reorganization of the helices association. These results are discussed in light of several proposed models of the membrane-bound colicin and channel structures.
Highlights
Four disulfide bonds wereengineered into the pore- duction of intramolecular disulfide bonds in several proteins of forming domainof colicinA to probe the conformational known structure has become a new strategy to increase the changes associated with its membrane insertion and protein stability and to study the protein folding pathways channel formation
The first outcome of this study is that the colicin pore for
Pore-forming colicins belontgo the peculiar class of proteins mation can be divided into two distinct steps, a membrane which can adopt two, in principle, exclusive conformations: a insertion step anda step leading to a conductingchannel
Summary
With loadingbuffer (60m Tris-HC1, pH 6.82;% SDS; 10% glyceroaln)d run on 7% SDS-polyacrylamide gelelectrophoresis, M urea. The W3110 strain containing the various mutated plasmids was grown incubated 5 min a t 37 "C in 100 nm sodium phosphate buffer (pH 7.2). Restriction enzymes were obtained by site-directed mutagenesis as pre- Insertion Kinetics of Colicin A into Phospholipid Vesicles-The viously described(Baty et al, 1987).ltventy oligonucleotides wereused quenching of the intrinsic fluorescence of colicin A by brominated dioto introduce 23 restriction sites. At time t = 0 the protein (AccYWMI,BstEII, ApaI, PpuMI, SplI, BarnHYNheYSacII, and MluI) is free and the fluorescence we observe is F,, whereas at t = m all the and treated as previously described to give E4. The observed fluoressingle strand of E4 was hybridized with six new oligonucleotides(XbaI, AurII, BspMII, SfiI, &I, and BstBI) and treated as described. Of paired oligonucleotides coding for W140C (H7) and G184C (H9b) between XhoI and PpuMI of the A150C (H8) single mutant and SacII
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