A very short peptide makes a voltage‐dependent ion channel: The critical length of the channel domain of colicin E1

Abstract

Cleavage of colicin E1 molecules with a variety of proteases or with cyanogen bromide (CNBr) generates COOH-terminal fragments which have channel-forming activity similar to that of intact colicin in planar lipid bilayer membranes. The smallest channel-forming fragment obtained by CNBr cleavage of the wild-type molecule consists of the C-terminal 152 amino acids. By the use of oligonucleotide-directed mutagenesis, we have made nine mutants along this 152 amino acid peptide, in which an amino acid was replaced by methionine in order to create a new CNBr cleavage site. The smallest of the CNBr-cleaved C-terminal fragments with channel-forming activity, in planar bilayer membranes, was generated by cleavage at new Met position 428 and has 94 amino acids, whereas a 75 amino acid peptide produced by cleavage of a new Met at position 447 did not have channel activity. The NH2-terminus of the channel-forming domain of colicin E1 appears therefore to lie between residues 428 and 447. Since, however, the last six C-terminal residues of the colicin can be removed without changing activity, the number of amino acids necessary to form the channel is 88 or less. In addition, the unique Cys residue in colicin E1 was replaced by Gly, and nine mutants were then made with Cys placed at sequential locations along the peptide for eventual use as sulfhydryl attachment sites to determine the local environment of the replaced amino acid. In the course of making 21 mutants, eight charged residues have been replaced by uncharged Met or Cys without changing the biological activity of the intact molecule. It has been proposed previously that the conformation of the colicin E1 channel is a barrel formed from five or six alpha-helices, each having 20 amino acids spanning the membrane and two to four residues making the turn at the boundary of the membrane. Our finding that 88 amino acids can make an active channel, combined with recently reported stoichiometric evidence that the channel is a monomer excludes this model and adds significant constraints which can be used in building a molecular model of the channel.

Translocation of inserted foreign epitopes by a channelforming protein

“…Western blot analysis with anti-HA antibody, for mutants II and III, or with anti-FLAG, for mutants IV and V, revealed that the truncated polypeptides were missing the C-terminal end, including the HA or FLAG epitope. Because C-terminal deletions eliminate the channelforming and bactericidal activities of channel-forming colicins, the truncated polypeptides should not have affected our measurements (23,24). Mutant HA-I, in contrast, was not degraded.…”

Calorimetric Investigations of the Structural Stability and Interactions of Colicin B Domains in Aqueous Solution and in the Presence of Phospholipid Bilayers

“…Although the isolated C-terminal fragment by itself is competent to insert into membranes (16), and pore-forming domains of other colicins exhibit pore-forming activities in vitro (17,(43)(44)(45), all three domains are required for recognition, translocation, and pore formation at intact cells in vivo. It appears that mutual interactions between different regions of colicin B help to maintain the translocation and the poreforming domains in less stable but functionally competent conformations.…”

A 136‐amino‐acid‐residue COOH‐terminal fragment of colicin A is endowed with ionophoric activity

“…The colicin El COOHterminal domain is significantly similar to colicin A [1,5 -71. The minimum size of this domain that is competent for channel function is under debate [8]. However, it is clear that a colicin El COOH-terminal fragment of 152 residues is competent for channel function [9].…”