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Rheostat Re-Wired: Alternative Hypotheses for the Control of Thioredoxin Reduction Potentials

Thioredoxins are small soluble proteins that contain a redox-active disulfide (CXXC). These disulfides are tuned to oxidizing or reducing potentials depending on the function of the thioredoxin within the cell. The mechanism by which the potential is tuned has been controversial, with two main hypot...

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Autores principales: Bewley, Kathryn D., Dey, Mishtu, Bjork, Rebekah E., Mitra, Sangha, Chobot, Sarah E., Drennan, Catherine L., Elliott, Sean J.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4395160/
https://www.ncbi.nlm.nih.gov/pubmed/25874934
http://dx.doi.org/10.1371/journal.pone.0122466
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author Bewley, Kathryn D.
Dey, Mishtu
Bjork, Rebekah E.
Mitra, Sangha
Chobot, Sarah E.
Drennan, Catherine L.
Elliott, Sean J.
author_facet Bewley, Kathryn D.
Dey, Mishtu
Bjork, Rebekah E.
Mitra, Sangha
Chobot, Sarah E.
Drennan, Catherine L.
Elliott, Sean J.
author_sort Bewley, Kathryn D.
collection PubMed
description Thioredoxins are small soluble proteins that contain a redox-active disulfide (CXXC). These disulfides are tuned to oxidizing or reducing potentials depending on the function of the thioredoxin within the cell. The mechanism by which the potential is tuned has been controversial, with two main hypotheses: first, that redox potential (E(m)) is specifically governed by a molecular ‘rheostat’—the XX amino acids, which influence the Cys pK(a) values, and thereby, E(m); and second, the overall thermodynamics of protein folding stability regulates the potential. Here, we use protein film voltammetry (PFV) to measure the pH dependence of the redox potentials of a series of wild-type and mutant archaeal Trxs, PFV and glutathionine-equilibrium to corroborate the measured potentials, the fluorescence probe BADAN to measure pK(a) values, guanidinium-based denaturation to measure protein unfolding, and X-ray crystallography to provide a structural basis for our functional analyses. We find that when these archaeal thioredoxins are probed directly using PFV, both the high and low potential thioredoxins display consistent 2H(+):2e(-) coupling over a physiological pH range, in conflict with the conventional ‘rheostat’ model. Instead, folding measurements reveals an excellent correlation to reduction potentials, supporting the second hypothesis and revealing the molecular mechanism of reduction potential control in the ubiquitous Trx family.
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spelling pubmed-43951602015-04-21 Rheostat Re-Wired: Alternative Hypotheses for the Control of Thioredoxin Reduction Potentials Bewley, Kathryn D. Dey, Mishtu Bjork, Rebekah E. Mitra, Sangha Chobot, Sarah E. Drennan, Catherine L. Elliott, Sean J. PLoS One Research Article Thioredoxins are small soluble proteins that contain a redox-active disulfide (CXXC). These disulfides are tuned to oxidizing or reducing potentials depending on the function of the thioredoxin within the cell. The mechanism by which the potential is tuned has been controversial, with two main hypotheses: first, that redox potential (E(m)) is specifically governed by a molecular ‘rheostat’—the XX amino acids, which influence the Cys pK(a) values, and thereby, E(m); and second, the overall thermodynamics of protein folding stability regulates the potential. Here, we use protein film voltammetry (PFV) to measure the pH dependence of the redox potentials of a series of wild-type and mutant archaeal Trxs, PFV and glutathionine-equilibrium to corroborate the measured potentials, the fluorescence probe BADAN to measure pK(a) values, guanidinium-based denaturation to measure protein unfolding, and X-ray crystallography to provide a structural basis for our functional analyses. We find that when these archaeal thioredoxins are probed directly using PFV, both the high and low potential thioredoxins display consistent 2H(+):2e(-) coupling over a physiological pH range, in conflict with the conventional ‘rheostat’ model. Instead, folding measurements reveals an excellent correlation to reduction potentials, supporting the second hypothesis and revealing the molecular mechanism of reduction potential control in the ubiquitous Trx family. Public Library of Science 2015-04-13 /pmc/articles/PMC4395160/ /pubmed/25874934 http://dx.doi.org/10.1371/journal.pone.0122466 Text en © 2015 Bewley et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Bewley, Kathryn D.
Dey, Mishtu
Bjork, Rebekah E.
Mitra, Sangha
Chobot, Sarah E.
Drennan, Catherine L.
Elliott, Sean J.
Rheostat Re-Wired: Alternative Hypotheses for the Control of Thioredoxin Reduction Potentials
title Rheostat Re-Wired: Alternative Hypotheses for the Control of Thioredoxin Reduction Potentials
title_full Rheostat Re-Wired: Alternative Hypotheses for the Control of Thioredoxin Reduction Potentials
title_fullStr Rheostat Re-Wired: Alternative Hypotheses for the Control of Thioredoxin Reduction Potentials
title_full_unstemmed Rheostat Re-Wired: Alternative Hypotheses for the Control of Thioredoxin Reduction Potentials
title_short Rheostat Re-Wired: Alternative Hypotheses for the Control of Thioredoxin Reduction Potentials
title_sort rheostat re-wired: alternative hypotheses for the control of thioredoxin reduction potentials
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4395160/
https://www.ncbi.nlm.nih.gov/pubmed/25874934
http://dx.doi.org/10.1371/journal.pone.0122466
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