Alteration of the pH- dependent ion selectivity of the colicin E1 channel by site-directed mutagenesis

Karen S. Jakes, Charles K. Abrams, Alan Finkelstein, Stephen L. Slatin

Research output: Contribution to journalArticle

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Abstract

Colicin E1 is a soluble, bacteriocidal protein that forms voltage-gated channels in planar lipid bilayers. The channel-forming region of the 522-amino acid protein is near the COOH terminus, and contains a 35-amino acid hydrophobic segment which is presumed to be important in interacting with the membrane. We have used site-directed mutagenesis in the region immediately upstream from the hydrophobic segment to construct several functional colicin mutants in which a wild-type residue was replaced with a cysteine. We also replaced the only naturally occurring cysteine in the molecule, Cys-505, with alanine, so that synthetically introduced cysteines could unambiguously serve as targets for chemical modification. All of the replacements reported here (at positions 449, 459, 473, 505, and some combinations) resulted in a channel that had an ion selectivity (K+ versus Cl-) identical to wild type at low pH. At higher pH, however, one of these mutations, which replaced the negatively charged aspartate at position 473 (the upstream boundary of the hydrophobic segment), resulted in a channel that was less cation-selective than was wild type. When the introduced Cys-473 was reacted with iodoacetic acid, which inserted a COOH group close to the position of the missing aspartate COOH, wild-type ion selectivity was restored, suggesting that the greater cation selectivity of the wild-type channel was directly produced by the negative charge at Asp-473. By comparing the ion selectivity of the Cys-473 mutant channel to that of the wild type as a function of the pH on the cis and trans sides of the membrane, it was possible to locate residue 473 close to the cis side. Locating in this manner the positions in the channel of particular residues places important constraints on channel model building.

Original languageEnglish (US)
Pages (from-to)6984-6991
Number of pages8
JournalJournal of Biological Chemistry
Volume265
Issue number12
StatePublished - 1990

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Colicins
Mutagenesis
Site-Directed Mutagenesis
Cysteine
Ions
Aspartic Acid
Cations
Iodoacetic Acid
Membranes
Amino Acids
Lipid bilayers
Chemical modification
Lipid Bilayers
Ion Channels
Alanine
Proteins
Mutation
Molecules
Electric potential

ASJC Scopus subject areas

  • Biochemistry

Cite this

Jakes, K. S., Abrams, C. K., Finkelstein, A., & Slatin, S. L. (1990). Alteration of the pH- dependent ion selectivity of the colicin E1 channel by site-directed mutagenesis. Journal of Biological Chemistry, 265(12), 6984-6991.

Alteration of the pH- dependent ion selectivity of the colicin E1 channel by site-directed mutagenesis. / Jakes, Karen S.; Abrams, Charles K.; Finkelstein, Alan; Slatin, Stephen L.

In: Journal of Biological Chemistry, Vol. 265, No. 12, 1990, p. 6984-6991.

Research output: Contribution to journalArticle

Jakes, KS, Abrams, CK, Finkelstein, A & Slatin, SL 1990, 'Alteration of the pH- dependent ion selectivity of the colicin E1 channel by site-directed mutagenesis', Journal of Biological Chemistry, vol. 265, no. 12, pp. 6984-6991.
Jakes, Karen S. ; Abrams, Charles K. ; Finkelstein, Alan ; Slatin, Stephen L. / Alteration of the pH- dependent ion selectivity of the colicin E1 channel by site-directed mutagenesis. In: Journal of Biological Chemistry. 1990 ; Vol. 265, No. 12. pp. 6984-6991.
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