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Protein Translocation across Planar Bilayers by the Colicin Ia Channel-Forming Domain: Where Will It End?
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 form...
Autores principales: | , , |
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Formato: | Texto |
Lenguaje: | English |
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The Rockefeller University Press
2000
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2230624/ https://www.ncbi.nlm.nih.gov/pubmed/11004207 |
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author | Kienker, Paul K. Jakes, Karen S. Finkelstein, Alan |
author_facet | Kienker, Paul K. Jakes, Karen S. Finkelstein, Alan |
author_sort | Kienker, Paul K. |
collection | PubMed |
description | 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. |
format | Text |
id | pubmed-2230624 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2000 |
publisher | The Rockefeller University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-22306242008-04-21 Protein Translocation across Planar Bilayers by the Colicin Ia Channel-Forming Domain: Where Will It End? Kienker, Paul K. Jakes, Karen S. Finkelstein, Alan J Gen Physiol Original Article 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. The Rockefeller University Press 2000-10-01 /pmc/articles/PMC2230624/ /pubmed/11004207 Text en © 2000 The Rockefeller University Press This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/). |
spellingShingle | Original Article Kienker, Paul K. Jakes, Karen S. Finkelstein, Alan Protein Translocation across Planar Bilayers by the Colicin Ia Channel-Forming Domain: Where Will It End? |
title | Protein Translocation across Planar Bilayers by the Colicin Ia Channel-Forming Domain: Where Will It End? |
title_full | Protein Translocation across Planar Bilayers by the Colicin Ia Channel-Forming Domain: Where Will It End? |
title_fullStr | Protein Translocation across Planar Bilayers by the Colicin Ia Channel-Forming Domain: Where Will It End? |
title_full_unstemmed | Protein Translocation across Planar Bilayers by the Colicin Ia Channel-Forming Domain: Where Will It End? |
title_short | Protein Translocation across Planar Bilayers by the Colicin Ia Channel-Forming Domain: Where Will It End? |
title_sort | protein translocation across planar bilayers by the colicin ia channel-forming domain: where will it end? |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2230624/ https://www.ncbi.nlm.nih.gov/pubmed/11004207 |
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