title: Chemical modification of the two histidine and single cysteine residues in the channel-forming domain of colicin E1.
abstract: The two histidine residues of COOH-terminal channel-forming peptides of colicin E1 were modified by addition of a carbethoxy group through pretreatment with diethylpyrocarbonate. The consequences of the modification were examined by the action of the altered product on both phospholipid vesicles and planar membranes. At pH 6, where activity is low, histidine modification resulted in a decrease of the single channel conductance from 20 pS to approximately 9 pS and a decrease in the selectivity for sodium relative to chloride, showing that histidine modification affected the permeability properties of the channel. At pH 4, where activity is high, the single channel conductance and ion selectivity were not significantly altered by histidine modification. The histidine modification assayed at pH 4 resulted in a threefold increase in the rate of Cl- efflux from asolectin vesicles, and a similar increase in conductance assayed with planar membranes. This conductance increase was inferred to arise from an increase in the fraction of bound histidine-modified colicin molecules forming channels at pH 4, since the increase in activity was not due to an increase in binding of the modified peptide, a change in ion selectivity, a change of single channel conductance, or a change in the pH dependence of binding. The sole cysteine in the colicin molecule was modified in 6 M urea with 5,5'-dithiobis(2-nitrobenzoic acid). The activities of the colicin and its COOH-terminal tryptic peptide were found to be unaffected by cysteine modification, arguing against a role of (-SH) groups in protein insertion and/or channel formation.
mesh_terms: D000595:Amino Acid Sequence; D003087:Colicins; D003545:Cysteine; D004553:Electric Conductivity; D004926:Escherichia coli; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007473:Ion Channels; D008564:Membrane Potentials; D010446:Peptide Fragments; D014357:Trypsin
chemical_list: D003087:Colicins; D007473:Ion Channels; D010446:Peptide Fragments; D006639:Histidine; D014357:Trypsin; D003545:Cysteine