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Chromophore binding to two cysteines increases quantum yield of near-infrared fluorescent proteins

Phytochromes are red/far-red light sensing photoreceptors employing linear tetrapyrroles as chromophores, which are covalently bound to a cysteine (Cys) residue in the chromophore-binding domain (CBD, composed of a PAS and a GAF domain). Recently, near-infrared (NIR) fluorescent proteins (FPs) engin...

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Autores principales: Buhrke, David, Tavraz, Neslihan N., Shcherbakova, Daria M., Sauthof, Luisa, Moldenhauer, Marcus, Vélazquez Escobar, Francisco, Verkhusha, Vladislav V., Hildebrandt, Peter, Friedrich, Thomas
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6372600/
https://www.ncbi.nlm.nih.gov/pubmed/30755663
http://dx.doi.org/10.1038/s41598-018-38433-2
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author Buhrke, David
Tavraz, Neslihan N.
Shcherbakova, Daria M.
Sauthof, Luisa
Moldenhauer, Marcus
Vélazquez Escobar, Francisco
Verkhusha, Vladislav V.
Hildebrandt, Peter
Friedrich, Thomas
author_facet Buhrke, David
Tavraz, Neslihan N.
Shcherbakova, Daria M.
Sauthof, Luisa
Moldenhauer, Marcus
Vélazquez Escobar, Francisco
Verkhusha, Vladislav V.
Hildebrandt, Peter
Friedrich, Thomas
author_sort Buhrke, David
collection PubMed
description Phytochromes are red/far-red light sensing photoreceptors employing linear tetrapyrroles as chromophores, which are covalently bound to a cysteine (Cys) residue in the chromophore-binding domain (CBD, composed of a PAS and a GAF domain). Recently, near-infrared (NIR) fluorescent proteins (FPs) engineered from bacterial phytochromes binding biliverdin IXα (BV), such as the iRFP series, have become invaluable probes for multicolor fluorescence microscopy and in vivo imaging. However, all current NIR FPs suffer from relatively low brightness. Here, by combining biochemical, spectroscopic and resonance Raman (RR) assays, we purified and characterized an iRFP variant that contains a BV chromophore simultaneously bound to two cysteines. This protein with the unusual double-Cys attached BV showed the highest fluorescence quantum yield (FQY) of 16.6% reported for NIR FPs, whereas the initial iRFP appeared to be a mixture of species with a mean FQY of 11.1%. The purified protein was also characterized with 1.3-fold higher extinction coefficient that together with FQY resulted in almost two-fold brighter fluorescence than the original iRFP as isolated. This work shows that the high FQY of iRFPs with two cysteines is a direct consequence of the double attachment. The PAS-Cys, GAF-Cys and double-Cys attachment each entails distinct configurational constraints of the BV adduct, which can be identified by distinct RR spectroscopic features, i.e. the marker band including the C=C stretching coordinate of the ring A-B methine bridge, which was previously identified as being characteristic for rigid chromophore embedment and high FQY. Our findings can be used to rationally engineer iRFP variants with enhanced FQYs.
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spelling pubmed-63726002019-02-19 Chromophore binding to two cysteines increases quantum yield of near-infrared fluorescent proteins Buhrke, David Tavraz, Neslihan N. Shcherbakova, Daria M. Sauthof, Luisa Moldenhauer, Marcus Vélazquez Escobar, Francisco Verkhusha, Vladislav V. Hildebrandt, Peter Friedrich, Thomas Sci Rep Article Phytochromes are red/far-red light sensing photoreceptors employing linear tetrapyrroles as chromophores, which are covalently bound to a cysteine (Cys) residue in the chromophore-binding domain (CBD, composed of a PAS and a GAF domain). Recently, near-infrared (NIR) fluorescent proteins (FPs) engineered from bacterial phytochromes binding biliverdin IXα (BV), such as the iRFP series, have become invaluable probes for multicolor fluorescence microscopy and in vivo imaging. However, all current NIR FPs suffer from relatively low brightness. Here, by combining biochemical, spectroscopic and resonance Raman (RR) assays, we purified and characterized an iRFP variant that contains a BV chromophore simultaneously bound to two cysteines. This protein with the unusual double-Cys attached BV showed the highest fluorescence quantum yield (FQY) of 16.6% reported for NIR FPs, whereas the initial iRFP appeared to be a mixture of species with a mean FQY of 11.1%. The purified protein was also characterized with 1.3-fold higher extinction coefficient that together with FQY resulted in almost two-fold brighter fluorescence than the original iRFP as isolated. This work shows that the high FQY of iRFPs with two cysteines is a direct consequence of the double attachment. The PAS-Cys, GAF-Cys and double-Cys attachment each entails distinct configurational constraints of the BV adduct, which can be identified by distinct RR spectroscopic features, i.e. the marker band including the C=C stretching coordinate of the ring A-B methine bridge, which was previously identified as being characteristic for rigid chromophore embedment and high FQY. Our findings can be used to rationally engineer iRFP variants with enhanced FQYs. Nature Publishing Group UK 2019-02-12 /pmc/articles/PMC6372600/ /pubmed/30755663 http://dx.doi.org/10.1038/s41598-018-38433-2 Text en © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Buhrke, David
Tavraz, Neslihan N.
Shcherbakova, Daria M.
Sauthof, Luisa
Moldenhauer, Marcus
Vélazquez Escobar, Francisco
Verkhusha, Vladislav V.
Hildebrandt, Peter
Friedrich, Thomas
Chromophore binding to two cysteines increases quantum yield of near-infrared fluorescent proteins
title Chromophore binding to two cysteines increases quantum yield of near-infrared fluorescent proteins
title_full Chromophore binding to two cysteines increases quantum yield of near-infrared fluorescent proteins
title_fullStr Chromophore binding to two cysteines increases quantum yield of near-infrared fluorescent proteins
title_full_unstemmed Chromophore binding to two cysteines increases quantum yield of near-infrared fluorescent proteins
title_short Chromophore binding to two cysteines increases quantum yield of near-infrared fluorescent proteins
title_sort chromophore binding to two cysteines increases quantum yield of near-infrared fluorescent proteins
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6372600/
https://www.ncbi.nlm.nih.gov/pubmed/30755663
http://dx.doi.org/10.1038/s41598-018-38433-2
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