On the explanation of the acidic pH requirement for in vitro activity of colicin E1. Site-directed mutagenesis at Glu-468.
The in vitro acidic pH dependence of colicin E1 channel activity was investigated by directed mutagenesis of Glu-468 in the colicin E1 channel domain, a residue conserved in the sequences of the four channel-forming colicins examined so far. Mutations were made to the amino acids leucine, serine, glutamine, or lysine, residues of different polarity and charge. All of the mutant polypeptides possessed high cytotoxic activity in vivo, although in vitro activity, especially with planar membranes, was lower than that of the wild type protein. A change in the in vitro acidic pH dependence of activity could be readily detected in the mutation to the hydrophobic leucine residue. The dependence of mutant activity on pH in the interval 3.5-5.0 was markedly smaller than that of the wild type, whether assayed on membrane vesicles or membrane bilayers. Differences in pH dependence between the wild type and the polar serine and glutamine mutants were small or of marginal statistical significance. No change in pH dependence could be detected with the charged lysine mutant. The residual pH dependence in all cases indicated that more than one carboxylic residue must be protonated to account for the increased activity at acidic pH values. A role of Glu-468 in the mechanism of channel formation or function was implied by the changes determined in vitro of channel parameters relative to the wild type: (i) the relatively small rates of current increase measured for colicin COOH-terminal peptide derived from the mutants, (ii) the small values of steady-state conductance of mutant peptide at pH 3.5, and (iii) the reduced anion selectivity of peptide from the serine mutant.
D000595:Amino Acid Sequence; D001483:Base Sequence; D003087:Colicins / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D005971:Glutamates; D018698:Glutamic Acid; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels / Q000187:drug effects; D008051:Lipid Bilayers; D008081:Liposomes; D008969:Molecular Sequence Data; D009154:Mutation; D010446:Peptide Fragments / Q000032:analysis; D010713:Phosphatidylcholines; D010743:Phospholipids; D010957:Plasmids
D003087:Colicins; D005971:Glutamates; D007473:Ion Channels; D008051:Lipid Bilayers; D008081:Liposomes; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D010743:Phospholipids; D018698:Glutamic Acid; C024990:asolectin