Protein translocation across planar bilayers by the colicin Ia channel-forming domain: Where will it end?

Paul K. Kienker, Karen S. Jakes, Alan Finkelstein

Research output: Contribution to journalArticlepeer-review

32 Scopus citations

Abstract

Colicin Ia, a 626-residue bactericidal protein, consists of three domains, with the carboxy-terminal domain (C domain) responsible for channel formation. Whole colicin Ia or C domain added to a planar lipid bilayer membrane forms voltage-gated channels. We have shown previously that the channel formed by whole colicin Ia has four membrane-spanning segments and an ~68-residue segment translocated across the membrane. Various experimental interventions could cause a longer or shorter segment within the C domain to be translocated, making us wonder why translocation normally stops where it does, near the amino-terminal end of the C domain (approximately residue 450). We hypothesized that regions upstream from the C domain prevent its amino-terminal end from moving into and across the membrane. To test this idea, we prepared C domain with a ligand attached near its amino terminus, added it to one side of a planar bilayer to form channels, and then probed from the opposite side with a water-soluble protein that can specifically bind the ligand. The binding of the probe had a dramatic effect on channel gating, demonstrating that the ligand (and hence the amino-terminal end of the C domain) had moved across the membrane. Experiments with larger colicin Ia fragments showed that a region of more than 165 residues, upstream from the C domain, can also move across the membrane. All of the colicin Ia carboxy-terminal fragments that we examined form channels that pass from a state of relatively normal conductance to a low-conductance state; we interpret this passage as a transition from a channel with four membrane-spanning segments to one with only three.

Original languageEnglish (US)
Pages (from-to)587-597
Number of pages11
JournalJournal of General Physiology
Volume116
Issue number4
DOIs
StatePublished - 2000

Keywords

  • His-tag antibody
  • Single-channel conductance
  • Streptavidin
  • Trypsin
  • Voltage-gated channels

ASJC Scopus subject areas

  • Physiology

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