Summary
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.
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Bullock, J.O., Cohen, F.S., Dankert, J.R., Cramer, W.A. 1983. Comparison of the macroscopic and single channel conductance properties of colicin E1 and its COOH-terminal tryptic peptide.J. Biol. Chem. 258:9908–9912
Burstein, Y., Walsh, K.A., Neurath, H. 1974. Evidence for an essential histidine residue in thermolysin.Biochemistry 13:205–210
Chan, P.T., Ohmori, H., Tomizawa, J., Lebowitz, J. 1985. Nucleotide sequence and gene organization of colicin E1.J. Biol. Chem. 260:8925–8935
Cleveland, M., Slatin, S., Finkelstein, A., Levinthal, C. 1983. Structure-function relationships for a voltage-dependent ion channel. Properties of COOH-terminal fragments of colicin E1.Proc. Natl. Acad. Sci. USA 80:3706–3710
Cramer, W.A., Dankert, J.R., Uratani, Y. 1983. The membrane channel-forming bactericidal protein, colicin E1.Biochim. Biophys. Acta 737:173–193
Dankert, J.R., Uratani, Y., Grabau, C., Cramer, W.A., Hermodson, M. 1982. On a domain structure of colicin E1. A COOH-terminal peptide fragment active in membrane depolarization.J. Biol. Chem. 257:3857–3863
Davidson, V.L., Brunden, K.R., Cramer, W.A., Cohen, F.S. 1984. Studies on the mechanism of action of channel-forming colicins using artificial membranes.J. Membrane Biol. 79:105–118
Davidson, V.L., Brunden, K.R., Cramer, W.A. 1985. Acidic pH requirement for insertion of colicin E1 into artificial membrane vesicles: Relevance to the mechanism of action of colicins and certain toxins.Proc. Natl. Acad. Sci. USA 82:1386–1390
Davidson, V.L., Cramer, W.A., Bishop, L.J., Brunden, K.R. 1984b. Dependence of the activity of colicin E1 in artificial membrane vesicles on pH, membrane potential, and vesicle size.J. Biol. Chem. 259:594–600
Dickenson, C.J., Dickinson, F.M. 1975. The role of an essential histidine residue of yeast alcohol dehydrogenase.Eur. J. Biochem. 52:595–603
Fong, C.T.O., Silver, L., Christman, D.R., Schwartz, I.L. 1960. On the mechanism of action of the antidiuretic hormone (vasopressin).Proc. Natl. Acad. Sci. USA 46:1273–1277
Holbrook, J.J., Ingram, V.E. 1973. Ionic properties of an essential histidine residue in pig heart lactate dehydrogenase.Biochem. J. 131:729–738
Kagawa, Y., Racker, E. 1971. Partial resolution of the enzymes catalyzing oxidative phosphorylation.J. Biol. Chem. 246: 5477–5487
Luria, S.E., Suit, J.L. 1982. Transmembrane channels produced by colicin molecules.In: Membranes and Transport. A. Martonosi, editor. Vol. 2, pp. 279–283. Plenum, New York
Means, G.E., Feeney, R.E. 1971. Chemical Modification of Proteins. p. 220. Holder-Day, San Francisco
Miles, E.W. 1977. Modification of histidyl residues in proteins by diethylpyrocarbonate.Methods Enzymol. 47:431–442
Montal, M., Mueller, P. 1972. Formation of bimolecular membranes from lipid monolayers and a study of their electrical properties.Proc. Natl. Acad. Sci. USA 69:3561–3566
Pattus, F., Heitz, F., Martinez, C., Provencher, S.W., Lazdunski, C. 1985. Secondary structure of the pore-forming colicin A and its C-terminal fragment. Experimental fact and structure prediction.Eur. J. Biochem. 152:681–689
Raymond, L., Slatin, S.L., Finkelstein, A. 1985. Channels formed by colicin E1 in planar lipid bilayers are large and exhibit pH-dependent ion selectivity.J. Membrane Biol. 84:173–181
Schwartz, S.A., Helinski, D.R. 1971. Purification and characterization of colicin E1.J. Biol. Chem. 246:6318–6327
Shiver, J.W., Bishop, L.J., deJong, P.J., Cohen, F.S., Cramer, W.A. 1986. E468→(Ser or Leu) mutation reduces the pH dependence of colicin E1 in vitro activity.Fed. Proc. 44:1794
Wright, H.T., Marston, A.W., Goldstein, D.J. 1984. A functional role for cysteine disulfides in the transmembrane transport of diphtheria toxin.J. Biol. Chem. 259:1649–1654
Yamada, M., Ebina, Y., Miyata, T., Nakazawa, T., Nakazawa, A. 1982. Nucleotide sequence of the structural gene for colicin E1 and predicted structure of the protein.Proc. Natl. Acad. Sci. USA 79:2827–2831
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Bishop, L.J., Cohen, F.S., Davidson, V.L. et al. Chemical modification of the two histidine and single cysteine residues in the channel-forming domain of colicin E1. J. Membrain Biol. 92, 237–245 (1986). https://doi.org/10.1007/BF01869392
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DOI: https://doi.org/10.1007/BF01869392