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Characterization of Fluorescent Proteins with Intramolecular Photostabilization

Genetically encodable fluorescent proteins have revolutionized biological imaging in vivo and in vitro. Despite their importance, their photophysical properties, i. e., brightness, count‐rate and photostability, are relatively poor compared to synthetic organic fluorophores or quantum dots. Intramol...

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Autores principales: Henrikus, Sarah S., Tassis, Konstantinos, Zhang, Lei, van der Velde, Jasper H. M., Gebhardt, Christian, Herrmann, Andreas, Jung, Gregor, Cordes, Thorben
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9291837/
https://www.ncbi.nlm.nih.gov/pubmed/34296494
http://dx.doi.org/10.1002/cbic.202100276
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author Henrikus, Sarah S.
Tassis, Konstantinos
Zhang, Lei
van der Velde, Jasper H. M.
Gebhardt, Christian
Herrmann, Andreas
Jung, Gregor
Cordes, Thorben
author_facet Henrikus, Sarah S.
Tassis, Konstantinos
Zhang, Lei
van der Velde, Jasper H. M.
Gebhardt, Christian
Herrmann, Andreas
Jung, Gregor
Cordes, Thorben
author_sort Henrikus, Sarah S.
collection PubMed
description Genetically encodable fluorescent proteins have revolutionized biological imaging in vivo and in vitro. Despite their importance, their photophysical properties, i. e., brightness, count‐rate and photostability, are relatively poor compared to synthetic organic fluorophores or quantum dots. Intramolecular photostabilizers were recently rediscovered as an effective approach to improve photophysical properties of organic fluorophores. Here, direct conjugation of triplet‐state quenchers or redox‐active substances creates high local concentrations of photostabilizer around the fluorophore. In this paper, we screen for effects of covalently linked photostabilizers on fluorescent proteins. We produced a double cysteine mutant (A206C/L221C) of α‐GFP for attachment of photostabilizer‐maleimides on the β‐barrel near the chromophore. Whereas labelling with photostabilizers such as trolox, a nitrophenyl group, and cyclooctatetraene, which are often used for organic fluorophores, had no effect on α‐GFP‐photostability, a substantial increase of photostability was found upon conjugation to azobenzene. Although the mechanism of the photostabilizing effects remains to be elucidated, we speculate that the higher triplet‐energy of azobenzene might be crucial for triplet‐quenching of fluorophores in the blue spectral range. Our study paves the way for the development of fluorescent proteins with photostabilizers in the protein barrel by methods such as unnatural amino acid incorporation.
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spelling pubmed-92918372022-07-20 Characterization of Fluorescent Proteins with Intramolecular Photostabilization Henrikus, Sarah S. Tassis, Konstantinos Zhang, Lei van der Velde, Jasper H. M. Gebhardt, Christian Herrmann, Andreas Jung, Gregor Cordes, Thorben Chembiochem Full Papers Genetically encodable fluorescent proteins have revolutionized biological imaging in vivo and in vitro. Despite their importance, their photophysical properties, i. e., brightness, count‐rate and photostability, are relatively poor compared to synthetic organic fluorophores or quantum dots. Intramolecular photostabilizers were recently rediscovered as an effective approach to improve photophysical properties of organic fluorophores. Here, direct conjugation of triplet‐state quenchers or redox‐active substances creates high local concentrations of photostabilizer around the fluorophore. In this paper, we screen for effects of covalently linked photostabilizers on fluorescent proteins. We produced a double cysteine mutant (A206C/L221C) of α‐GFP for attachment of photostabilizer‐maleimides on the β‐barrel near the chromophore. Whereas labelling with photostabilizers such as trolox, a nitrophenyl group, and cyclooctatetraene, which are often used for organic fluorophores, had no effect on α‐GFP‐photostability, a substantial increase of photostability was found upon conjugation to azobenzene. Although the mechanism of the photostabilizing effects remains to be elucidated, we speculate that the higher triplet‐energy of azobenzene might be crucial for triplet‐quenching of fluorophores in the blue spectral range. Our study paves the way for the development of fluorescent proteins with photostabilizers in the protein barrel by methods such as unnatural amino acid incorporation. John Wiley and Sons Inc. 2021-07-22 2021-12-02 /pmc/articles/PMC9291837/ /pubmed/34296494 http://dx.doi.org/10.1002/cbic.202100276 Text en © 2021 The Authors. ChemBioChem published by Wiley-VCH GmbH https://creativecommons.org/licenses/by-nc/4.0/This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc/4.0/ (https://creativecommons.org/licenses/by-nc/4.0/) License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
spellingShingle Full Papers
Henrikus, Sarah S.
Tassis, Konstantinos
Zhang, Lei
van der Velde, Jasper H. M.
Gebhardt, Christian
Herrmann, Andreas
Jung, Gregor
Cordes, Thorben
Characterization of Fluorescent Proteins with Intramolecular Photostabilization
title Characterization of Fluorescent Proteins with Intramolecular Photostabilization
title_full Characterization of Fluorescent Proteins with Intramolecular Photostabilization
title_fullStr Characterization of Fluorescent Proteins with Intramolecular Photostabilization
title_full_unstemmed Characterization of Fluorescent Proteins with Intramolecular Photostabilization
title_short Characterization of Fluorescent Proteins with Intramolecular Photostabilization
title_sort characterization of fluorescent proteins with intramolecular photostabilization
topic Full Papers
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9291837/
https://www.ncbi.nlm.nih.gov/pubmed/34296494
http://dx.doi.org/10.1002/cbic.202100276
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