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Helix N-Cap Residues Drive the Acid Unfolding That Is Essential in the Action of the Toxin Colicin A

[Image: see text] Numerous bacterial toxins and other virulence factors use low pH as a trigger to convert from water-soluble to membrane-inserted states. In the case of colicins, the pore-forming domain of colicin A (ColA-P) has been shown both to undergo a clear acidic unfolding transition and to...

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Autores principales: Huang, Yan, Soliakov, Andrei, Le Brun, Anton P., Macdonald, Colin, Johnson, Christopher L., Solovyova, Alexandra S., Waller, Helen, Moore, Geoffrey R., Lakey, Jeremy H.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Chemical Society 2019
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6899464/
https://www.ncbi.nlm.nih.gov/pubmed/31686499
http://dx.doi.org/10.1021/acs.biochem.9b00705
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author Huang, Yan
Soliakov, Andrei
Le Brun, Anton P.
Macdonald, Colin
Johnson, Christopher L.
Solovyova, Alexandra S.
Waller, Helen
Moore, Geoffrey R.
Lakey, Jeremy H.
author_facet Huang, Yan
Soliakov, Andrei
Le Brun, Anton P.
Macdonald, Colin
Johnson, Christopher L.
Solovyova, Alexandra S.
Waller, Helen
Moore, Geoffrey R.
Lakey, Jeremy H.
author_sort Huang, Yan
collection PubMed
description [Image: see text] Numerous bacterial toxins and other virulence factors use low pH as a trigger to convert from water-soluble to membrane-inserted states. In the case of colicins, the pore-forming domain of colicin A (ColA-P) has been shown both to undergo a clear acidic unfolding transition and to require acidic lipids in the cytoplasmic membrane, whereas its close homologue colicin N shows neither behavior. Compared to that of ColN-P, the ColA-P primary structure reveals the replacement of several uncharged residues with aspartyl residues, which upon replacement with alanine induce an unfolded state at neutral pH. Here we investigate ColA-P’s structural requirement for these critical aspartyl residues that are largely situated at the N-termini of α helices. As previously shown in model peptides, the charged carboxylate side chain can act as a stabilizing helix N-Cap group by interacting with free amide hydrogen bond donors. Because this could explain ColA-P destabilization when the aspartyl residues are protonated or replaced with alanyl residues, we test the hypothesis by inserting asparagine, glutamine, and glutamate residues at these sites. We combine urea (fluorescence and circular dichroism) and thermal (circular dichroism and differential scanning calorimetry) denaturation experiments with (1)H–(15)N heteronuclear single-quantum coherence nuclear magnetic resonance spectroscopy of ColA-P at different pH values to provide a comprehensive description of the unfolding process and confirm the N-Cap hypothesis. Furthermore, we reveal that, in urea, the single domain ColA-P unfolds in two steps; low pH destabilizes the first step and stabilizes the second.
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spelling pubmed-68994642019-12-10 Helix N-Cap Residues Drive the Acid Unfolding That Is Essential in the Action of the Toxin Colicin A Huang, Yan Soliakov, Andrei Le Brun, Anton P. Macdonald, Colin Johnson, Christopher L. Solovyova, Alexandra S. Waller, Helen Moore, Geoffrey R. Lakey, Jeremy H. Biochemistry [Image: see text] Numerous bacterial toxins and other virulence factors use low pH as a trigger to convert from water-soluble to membrane-inserted states. In the case of colicins, the pore-forming domain of colicin A (ColA-P) has been shown both to undergo a clear acidic unfolding transition and to require acidic lipids in the cytoplasmic membrane, whereas its close homologue colicin N shows neither behavior. Compared to that of ColN-P, the ColA-P primary structure reveals the replacement of several uncharged residues with aspartyl residues, which upon replacement with alanine induce an unfolded state at neutral pH. Here we investigate ColA-P’s structural requirement for these critical aspartyl residues that are largely situated at the N-termini of α helices. As previously shown in model peptides, the charged carboxylate side chain can act as a stabilizing helix N-Cap group by interacting with free amide hydrogen bond donors. Because this could explain ColA-P destabilization when the aspartyl residues are protonated or replaced with alanyl residues, we test the hypothesis by inserting asparagine, glutamine, and glutamate residues at these sites. We combine urea (fluorescence and circular dichroism) and thermal (circular dichroism and differential scanning calorimetry) denaturation experiments with (1)H–(15)N heteronuclear single-quantum coherence nuclear magnetic resonance spectroscopy of ColA-P at different pH values to provide a comprehensive description of the unfolding process and confirm the N-Cap hypothesis. Furthermore, we reveal that, in urea, the single domain ColA-P unfolds in two steps; low pH destabilizes the first step and stabilizes the second. American Chemical Society 2019-11-05 2019-12-03 /pmc/articles/PMC6899464/ /pubmed/31686499 http://dx.doi.org/10.1021/acs.biochem.9b00705 Text en Copyright © 2019 American Chemical Society This is an open access article published under a Creative Commons Attribution (CC-BY) License (http://pubs.acs.org/page/policy/authorchoice_ccby_termsofuse.html) , which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited.
spellingShingle Huang, Yan
Soliakov, Andrei
Le Brun, Anton P.
Macdonald, Colin
Johnson, Christopher L.
Solovyova, Alexandra S.
Waller, Helen
Moore, Geoffrey R.
Lakey, Jeremy H.
Helix N-Cap Residues Drive the Acid Unfolding That Is Essential in the Action of the Toxin Colicin A
title Helix N-Cap Residues Drive the Acid Unfolding That Is Essential in the Action of the Toxin Colicin A
title_full Helix N-Cap Residues Drive the Acid Unfolding That Is Essential in the Action of the Toxin Colicin A
title_fullStr Helix N-Cap Residues Drive the Acid Unfolding That Is Essential in the Action of the Toxin Colicin A
title_full_unstemmed Helix N-Cap Residues Drive the Acid Unfolding That Is Essential in the Action of the Toxin Colicin A
title_short Helix N-Cap Residues Drive the Acid Unfolding That Is Essential in the Action of the Toxin Colicin A
title_sort helix n-cap residues drive the acid unfolding that is essential in the action of the toxin colicin a
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6899464/
https://www.ncbi.nlm.nih.gov/pubmed/31686499
http://dx.doi.org/10.1021/acs.biochem.9b00705
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