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Structure and Function of Redox-Sensitive Superfolder Green Fluorescent Protein Variant
AIMS: Genetically encoded green fluorescent protein (GFP)-based redox biosensors are widely used to monitor specific and dynamic redox processes in living cells. Over the last few years, various biosensors for a variety of applications were engineered and enhanced to match the organism and cellular...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Mary Ann Liebert, Inc., publishers
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9293687/ https://www.ncbi.nlm.nih.gov/pubmed/35072524 http://dx.doi.org/10.1089/ars.2021.0234 |
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author | Heimsch, Kim C. Gertzen, Christoph G.W. Schuh, Anna Katharina Nietzel, Thomas Rahlfs, Stefan Przyborski, Jude M. Gohlke, Holger Schwarzländer, Markus Becker, Katja Fritz-Wolf, Karin |
author_facet | Heimsch, Kim C. Gertzen, Christoph G.W. Schuh, Anna Katharina Nietzel, Thomas Rahlfs, Stefan Przyborski, Jude M. Gohlke, Holger Schwarzländer, Markus Becker, Katja Fritz-Wolf, Karin |
author_sort | Heimsch, Kim C. |
collection | PubMed |
description | AIMS: Genetically encoded green fluorescent protein (GFP)-based redox biosensors are widely used to monitor specific and dynamic redox processes in living cells. Over the last few years, various biosensors for a variety of applications were engineered and enhanced to match the organism and cellular environments, which should be investigated. In this context, the unicellular intraerythrocytic parasite Plasmodium, the causative agent of malaria, represents a challenge, as the small size of the organism results in weak fluorescence signals that complicate precise measurements, especially for cell compartment-specific observations. To address this, we have functionally and structurally characterized an enhanced redox biosensor superfolder roGFP2 (sfroGFP2). RESULTS: SfroGFP2 retains roGFP2-like behavior, yet with improved fluorescence intensity (FI) in cellulo. SfroGFP2-based redox biosensors are pH insensitive in a physiological pH range and show midpoint potentials comparable with roGFP2-based redox biosensors. Using crystallography and rigidity theory, we identified the superfolding mutations as being responsible for improved structural stability of the biosensor in a redox-sensitive environment, thus explaining the improved FI in cellulo. INNOVATION: This work provides insight into the structure and function of GFP-based redox biosensors. It describes an improved redox biosensor (sfroGFP2) suitable for measuring oxidizing effects within small cells where applicability of other redox sensor variants is limited. CONCLUSION: Improved structural stability of sfroGFP2 gives rise to increased FI in cellulo. Fusion to hGrx1 (human glutaredoxin-1) provides the hitherto most suitable biosensor for measuring oxidizing effects in Plasmodium. This sensor is of major interest for studying glutathione redox changes in small cells, as well as subcellular compartments in general. Antioxid. Redox Signal. 37, 1–18. |
format | Online Article Text |
id | pubmed-9293687 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | Mary Ann Liebert, Inc., publishers |
record_format | MEDLINE/PubMed |
spelling | pubmed-92936872022-08-01 Structure and Function of Redox-Sensitive Superfolder Green Fluorescent Protein Variant Heimsch, Kim C. Gertzen, Christoph G.W. Schuh, Anna Katharina Nietzel, Thomas Rahlfs, Stefan Przyborski, Jude M. Gohlke, Holger Schwarzländer, Markus Becker, Katja Fritz-Wolf, Karin Antioxid Redox Signal VOLUME 37 NUMBER 1 JULY 2022 AIMS: Genetically encoded green fluorescent protein (GFP)-based redox biosensors are widely used to monitor specific and dynamic redox processes in living cells. Over the last few years, various biosensors for a variety of applications were engineered and enhanced to match the organism and cellular environments, which should be investigated. In this context, the unicellular intraerythrocytic parasite Plasmodium, the causative agent of malaria, represents a challenge, as the small size of the organism results in weak fluorescence signals that complicate precise measurements, especially for cell compartment-specific observations. To address this, we have functionally and structurally characterized an enhanced redox biosensor superfolder roGFP2 (sfroGFP2). RESULTS: SfroGFP2 retains roGFP2-like behavior, yet with improved fluorescence intensity (FI) in cellulo. SfroGFP2-based redox biosensors are pH insensitive in a physiological pH range and show midpoint potentials comparable with roGFP2-based redox biosensors. Using crystallography and rigidity theory, we identified the superfolding mutations as being responsible for improved structural stability of the biosensor in a redox-sensitive environment, thus explaining the improved FI in cellulo. INNOVATION: This work provides insight into the structure and function of GFP-based redox biosensors. It describes an improved redox biosensor (sfroGFP2) suitable for measuring oxidizing effects within small cells where applicability of other redox sensor variants is limited. CONCLUSION: Improved structural stability of sfroGFP2 gives rise to increased FI in cellulo. Fusion to hGrx1 (human glutaredoxin-1) provides the hitherto most suitable biosensor for measuring oxidizing effects in Plasmodium. This sensor is of major interest for studying glutathione redox changes in small cells, as well as subcellular compartments in general. Antioxid. Redox Signal. 37, 1–18. Mary Ann Liebert, Inc., publishers 2022-07-01 2022-07-08 /pmc/articles/PMC9293687/ /pubmed/35072524 http://dx.doi.org/10.1089/ars.2021.0234 Text en © Kim C. Heimsch et al., 2022; Published by Mary Ann Liebert, Inc. https://creativecommons.org/licenses/by-nc/4.0/This Open Access article is distributed under the terms of the Creative Commons Attribution Noncommercial License [CC-BY-NC] (http://creativecommons.org/licenses/by-nc/4.0/ (https://creativecommons.org/licenses/by-nc/4.0/) ) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and the source are cited. |
spellingShingle | VOLUME 37 NUMBER 1 JULY 2022 Heimsch, Kim C. Gertzen, Christoph G.W. Schuh, Anna Katharina Nietzel, Thomas Rahlfs, Stefan Przyborski, Jude M. Gohlke, Holger Schwarzländer, Markus Becker, Katja Fritz-Wolf, Karin Structure and Function of Redox-Sensitive Superfolder Green Fluorescent Protein Variant |
title | Structure and Function of Redox-Sensitive Superfolder Green Fluorescent Protein Variant |
title_full | Structure and Function of Redox-Sensitive Superfolder Green Fluorescent Protein Variant |
title_fullStr | Structure and Function of Redox-Sensitive Superfolder Green Fluorescent Protein Variant |
title_full_unstemmed | Structure and Function of Redox-Sensitive Superfolder Green Fluorescent Protein Variant |
title_short | Structure and Function of Redox-Sensitive Superfolder Green Fluorescent Protein Variant |
title_sort | structure and function of redox-sensitive superfolder green fluorescent protein variant |
topic | VOLUME 37 NUMBER 1 JULY 2022 |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9293687/ https://www.ncbi.nlm.nih.gov/pubmed/35072524 http://dx.doi.org/10.1089/ars.2021.0234 |
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