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Redox-sensitive GFP fusions for monitoring the catalytic mechanism and inactivation of peroxiredoxins in living cells

Redox-sensitive green fluorescent protein 2 (roGFP2) is a valuable tool for redox measurements in living cells. Here, we demonstrate that roGFP2 can also be used to gain mechanistic insights into redox catalysis in vivo. In vitro enzyme properties such as the rate-limiting reduction of wild type and...

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Autores principales: Staudacher, Verena, Trujillo, Madia, Diederichs, Tim, Dick, Tobias P., Radi, Rafael, Morgan, Bruce, Deponte, Marcel
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5684490/
https://www.ncbi.nlm.nih.gov/pubmed/29128826
http://dx.doi.org/10.1016/j.redox.2017.10.017
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author Staudacher, Verena
Trujillo, Madia
Diederichs, Tim
Dick, Tobias P.
Radi, Rafael
Morgan, Bruce
Deponte, Marcel
author_facet Staudacher, Verena
Trujillo, Madia
Diederichs, Tim
Dick, Tobias P.
Radi, Rafael
Morgan, Bruce
Deponte, Marcel
author_sort Staudacher, Verena
collection PubMed
description Redox-sensitive green fluorescent protein 2 (roGFP2) is a valuable tool for redox measurements in living cells. Here, we demonstrate that roGFP2 can also be used to gain mechanistic insights into redox catalysis in vivo. In vitro enzyme properties such as the rate-limiting reduction of wild type and mutant forms of the model peroxiredoxin PfAOP are shown to correlate with the ratiometrically measured degree of oxidation of corresponding roGFP2 fusion proteins. Furthermore, stopped-flow kinetic measurements of the oxidative half-reaction of PfAOP support the interpretation that changes in the roGFP2 signal can be used to map hyperoxidation-based inactivation of the attached peroxidase. Potential future applications of our system include the improvement of redox sensors, the estimation of absolute intracellular peroxide concentrations and the in vivo assessment of protein structure-function relationships that cannot easily be addressed with recombinant enzymes, for example, the effect of post-translational protein modifications on enzyme catalysis.
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spelling pubmed-56844902017-11-20 Redox-sensitive GFP fusions for monitoring the catalytic mechanism and inactivation of peroxiredoxins in living cells Staudacher, Verena Trujillo, Madia Diederichs, Tim Dick, Tobias P. Radi, Rafael Morgan, Bruce Deponte, Marcel Redox Biol Research Paper Redox-sensitive green fluorescent protein 2 (roGFP2) is a valuable tool for redox measurements in living cells. Here, we demonstrate that roGFP2 can also be used to gain mechanistic insights into redox catalysis in vivo. In vitro enzyme properties such as the rate-limiting reduction of wild type and mutant forms of the model peroxiredoxin PfAOP are shown to correlate with the ratiometrically measured degree of oxidation of corresponding roGFP2 fusion proteins. Furthermore, stopped-flow kinetic measurements of the oxidative half-reaction of PfAOP support the interpretation that changes in the roGFP2 signal can be used to map hyperoxidation-based inactivation of the attached peroxidase. Potential future applications of our system include the improvement of redox sensors, the estimation of absolute intracellular peroxide concentrations and the in vivo assessment of protein structure-function relationships that cannot easily be addressed with recombinant enzymes, for example, the effect of post-translational protein modifications on enzyme catalysis. Elsevier 2017-10-26 /pmc/articles/PMC5684490/ /pubmed/29128826 http://dx.doi.org/10.1016/j.redox.2017.10.017 Text en © 2017 The Authors http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Research Paper
Staudacher, Verena
Trujillo, Madia
Diederichs, Tim
Dick, Tobias P.
Radi, Rafael
Morgan, Bruce
Deponte, Marcel
Redox-sensitive GFP fusions for monitoring the catalytic mechanism and inactivation of peroxiredoxins in living cells
title Redox-sensitive GFP fusions for monitoring the catalytic mechanism and inactivation of peroxiredoxins in living cells
title_full Redox-sensitive GFP fusions for monitoring the catalytic mechanism and inactivation of peroxiredoxins in living cells
title_fullStr Redox-sensitive GFP fusions for monitoring the catalytic mechanism and inactivation of peroxiredoxins in living cells
title_full_unstemmed Redox-sensitive GFP fusions for monitoring the catalytic mechanism and inactivation of peroxiredoxins in living cells
title_short Redox-sensitive GFP fusions for monitoring the catalytic mechanism and inactivation of peroxiredoxins in living cells
title_sort redox-sensitive gfp fusions for monitoring the catalytic mechanism and inactivation of peroxiredoxins in living cells
topic Research Paper
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5684490/
https://www.ncbi.nlm.nih.gov/pubmed/29128826
http://dx.doi.org/10.1016/j.redox.2017.10.017
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