Chemical modification of the two histidine and single cysteine residues in the channel-forming domain of colicin E1
- 1 October 1986
- journal article
- research article
- Published by Springer Nature in The Journal of Membrane Biology
- Vol. 92 (3), 237-245
- https://doi.org/10.1007/bf01869392
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 (i) an increase in binding of the modified peptide, (ii) a change in ion selectivity, (iii) a change of single channel conductance, or (iv) a change in the pH dependence of binding. The sole cysteine in the colicin molecule was modified in 6m 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.Keywords
This publication has 27 references indexed in Scilit:
- Secondary structure of the pore‐forming colicin A and its C‐terminal fragmentEuropean Journal of Biochemistry, 1985
- Channels formed by colicin E1 in planar lipid bilayers are large and exhibit pH-dependent ion selectivityThe Journal of Membrane Biology, 1985
- Studies on the mechanism of action of channel-forming colicins using artificial membranesThe Journal of Membrane Biology, 1984
- Structure-function relationships for a voltage-dependent ion channel: properties of COOH-terminal fragments of colicin E1.Proceedings of the National Academy of Sciences, 1983
- The membrane channel-forming bacteriocidal protein, colicin ElBiochimica et Biophysica Acta (BBA) - Reviews on Biomembranes, 1983
- Nucleotide sequence of the structural gene for colicin E1 and predicted structure of the protein.Proceedings of the National Academy of Sciences, 1982
- [41] Modification of histidyl residues in proteins by diethylpyrocarbonateMethods in Enzymology, 1977
- The Role of an Essential Histidine Residue of Yeast Alcohol DehydrogenaseEuropean Journal of Biochemistry, 1975
- Evidence of an essential histidine residue in thermolysinBiochemistry, 1974
- ON THE MECHANISM OF ACTION OF THE ANTIDIURETIC HORMONE (VASOPRESSIN)Proceedings of the National Academy of Sciences, 1960