Identification of a translocated protein segment in a voltage-dependent channel

Nature. 1994 Sep 8;371(6493):158-61. doi: 10.1038/371158a0.


Voltage-gated channels undergo a conformational change in response to changes in transmembrane voltage. Here we use site-directed biotinylation to create conformation-sensitive sites on colicin Ia, a bacteriocidal protein that forms a voltage-sensitive membrane channel, which can be monitored by electrophysiological methods. We investigated a model of gating developed for the partly homologous colicin E1 that is based on the insertion of regions of the protein into the membrane in response to cis-positive voltages. Site-directed cysteine mutagenesis, followed by chemical modification, was used to attach a biotin molecule covalently to a series of unique sites on colicin Ia. The modified protein was incorporated into planar lipid membranes, where the introduced biotin moiety served as a site to bind the water-soluble protein streptavidin, added to one side of the membrane or the other. Our results show that colicin gating is associated with the translocation across the membrane of a segment of the protein of at least 31 amino acids.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / pharmacology
  • Biological Transport
  • Biotin
  • Colicins / chemistry
  • Colicins / drug effects
  • Colicins / genetics
  • Colicins / metabolism*
  • Cysteine / genetics
  • Cysteine / metabolism
  • Electrophysiology
  • Ion Channel Gating*
  • Ion Channels / chemistry
  • Ion Channels / drug effects
  • Ion Channels / genetics
  • Ion Channels / metabolism*
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Peptide Fragments / metabolism*
  • Protein Conformation
  • Streptavidin


  • Bacterial Proteins
  • Colicins
  • Ion Channels
  • Peptide Fragments
  • Biotin
  • Streptavidin
  • Cysteine