High level expression of His-tagged colicin pore-forming domains and reflections on the sites for pore formation in the inner membrane.

There exists ample evidence for the assumption that pore-forming colicins cannot exert their toxicity within the producing cell and that they must gain access to the outer face of the cytoplasmic membrane to achieve this. We wished to construct pET-vectors to produce pore-forming domains of colicin A and N with N-terminal hexa-histidine tags under the control of a T7 promoter. This was only possible when the correct immunity protein was also present. Hence it appears that this system exhibits the peculiarity that there is a toxicity associated with the over produced pore-forming domain. However, when the ratio of colicin to immunity protein is compared it is still clear that direct insertion into the cytoplasmic membrane does not occur and that membrane translocation of the colicin at limited sites may be occurring. This article reviews previous literature on the subject in terms of a model for limited sites of colicin action.

Surface aspartate residues are essential for the stability of colicin A P-domain: a mechanism for the formation of an acidic molten-globule.

The pore-forming domains of members of a family of bacterial toxins, colicins N and A, share > 50% sequence identity, identical folds and yet display strikingly different behavior in acid conditions. At low pH colicin A forms a molten globule state while colicin N retains a native fold. This is relevant to in vivo activity since colicin A requires acidic phospholipids for its toxic activity but colicin N does not. The pI of colicin A (5.25) is far lower than that of colicin N (10.2) because colicin A contains seven extra aspartate residues. We first introduced separately each of these acidic amino acids into homologous sites in colicin N, but none caused destabilization at low pH. However, in the reverse experiment, the sequential replacement of these acidic side chains of colicin A by alanine revealed six sites where this change destabilized the protein at neutral pH. Some of these residues, which each contribute less than 4% to the total negative charge, appear to stabilize the protein via a network of hydrogen bonds and charge pairs which are sensitive to protonation. Other residues have no clear interactions that explain their importance. The colicin A is thus a protein that relies upon acid sensitive interactions for its stability at neutral pH and its in vivo activity.