A very short peptide makes a voltage-dependent ion channel

the critical length of the channel domain of colicin E1.

Q. R. Liu, V. Crozel, F. Levinthal, S. Slatin, A. Finkelstein, C. Levinthal

Research output: Contribution to journalArticle

47 Citations (Scopus)

Abstract

Cleavage of colicin E1 molecules with a variety of proteases or with cyanogen bromide (CNBr) generates COOH-terminal fragments which have channel-forming activity similar to that of intact colicin in planar lipid bilayer membranes. The smallest channel-forming fragment obtained by CNBr cleavage of the wild-type molecule consists of the C-terminal 152 amino acids. By the use of oligonucleotide-directed mutagenesis, we have made nine mutants along this 152 amino acid peptide, in which an amino acid was replaced by methionine in order to create a new CNBr cleavage site. The smallest of the CNBr-cleaved C-terminal fragments with channel-forming activity, in planar bilayer membranes, was generated by cleavage at new Met position 428 and has 94 amino acids, whereas a 75 amino acid peptide produced by cleavage of a new Met at position 447 did not have channel activity. The NH2-terminus of the channel-forming domain of colicin E1 appears therefore to lie between residues 428 and 447. Since, however, the last six C-terminal residues of the colicin can be removed without changing activity, the number of amino acids necessary to form the channel is 88 or less. In addition, the unique Cys residue in colicin E1 was replaced by Gly, and nine mutants were then made with Cys placed at sequential locations along the peptide for eventual use as sulfhydryl attachment sites to determine the local environment of the replaced amino acid. In the course of making 21 mutants, eight charged residues have been replaced by uncharged Met or Cys without changing the biological activity of the intact molecule. It has been proposed previously that the conformation of the colicin E1 channel is a barrel formed from five or six alpha-helices, each having 20 amino acids spanning the membrane and two to four residues making the turn at the boundary of the membrane. Our finding that 88 amino acids can make an active channel, combined with recently reported stoichiometric evidence that the channel is a monomer excludes this model and adds significant constraints which can be used in building a molecular model of the channel.

Original languageEnglish (US)
Pages (from-to)218-229
Number of pages12
JournalProteins
Volume1
Issue number3
StatePublished - Nov 1986

Fingerprint

Colicins
Ion Channels
Amino Acids
Peptides
Cyanogen Bromide
Electric potential
Membranes
Molecules
Mutagenesis
Lipid bilayers
Molecular Models
Lipid Bilayers
Membrane Lipids
Site-Directed Mutagenesis
Bioactivity
Oligonucleotides
Methionine
Conformations
Peptide Hydrolases
Monomers

ASJC Scopus subject areas

  • Biochemistry
  • Genetics
  • Structural Biology

Cite this

Liu, Q. R., Crozel, V., Levinthal, F., Slatin, S., Finkelstein, A., & Levinthal, C. (1986). A very short peptide makes a voltage-dependent ion channel: the critical length of the channel domain of colicin E1. Proteins, 1(3), 218-229.

A very short peptide makes a voltage-dependent ion channel : the critical length of the channel domain of colicin E1. / Liu, Q. R.; Crozel, V.; Levinthal, F.; Slatin, S.; Finkelstein, A.; Levinthal, C.

In: Proteins, Vol. 1, No. 3, 11.1986, p. 218-229.

Research output: Contribution to journalArticle

Liu, QR, Crozel, V, Levinthal, F, Slatin, S, Finkelstein, A & Levinthal, C 1986, 'A very short peptide makes a voltage-dependent ion channel: the critical length of the channel domain of colicin E1.', Proteins, vol. 1, no. 3, pp. 218-229.
Liu QR, Crozel V, Levinthal F, Slatin S, Finkelstein A, Levinthal C. A very short peptide makes a voltage-dependent ion channel: the critical length of the channel domain of colicin E1. Proteins. 1986 Nov;1(3):218-229.
Liu, Q. R. ; Crozel, V. ; Levinthal, F. ; Slatin, S. ; Finkelstein, A. ; Levinthal, C. / A very short peptide makes a voltage-dependent ion channel : the critical length of the channel domain of colicin E1. In: Proteins. 1986 ; Vol. 1, No. 3. pp. 218-229.
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