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Impact of Double Covalent Binding of BV in NIR FPs on Their Spectral and Physicochemical Properties

Understanding the photophysical properties and stability of near-infrared fluorescent proteins (NIR FPs) based on bacterial phytochromes is of great importance for the design of efficient fluorescent probes for use in cells and in vivo. Previously, the natural ligand of NIR FPs biliverdin (BV) has b...

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Autores principales: Stepanenko, Olga V., Kuznetsova, Irina M., Turoverov, Konstantin K., Stepanenko, Olesya V.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9267011/
https://www.ncbi.nlm.nih.gov/pubmed/35806351
http://dx.doi.org/10.3390/ijms23137347
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author Stepanenko, Olga V.
Kuznetsova, Irina M.
Turoverov, Konstantin K.
Stepanenko, Olesya V.
author_facet Stepanenko, Olga V.
Kuznetsova, Irina M.
Turoverov, Konstantin K.
Stepanenko, Olesya V.
author_sort Stepanenko, Olga V.
collection PubMed
description Understanding the photophysical properties and stability of near-infrared fluorescent proteins (NIR FPs) based on bacterial phytochromes is of great importance for the design of efficient fluorescent probes for use in cells and in vivo. Previously, the natural ligand of NIR FPs biliverdin (BV) has been revealed to be capable of covalent binding to the inherent cysteine residue in the PAS domain (Cys15), and to the cysteine residue introduced into the GAF domain (Cys256), as well as simultaneously with these two residues. Here, based on the spectroscopic analysis of several NIR FPs with both cysteine residues in PAS and GAF domains, we show that the covalent binding of BV simultaneously with two domains is the reason for the higher quantum yield of BV fluorescence in these proteins as a result of rigid fixation of the chromophore in their chromophore-binding pocket. We demonstrate that since the attachment sites are located in different regions of the polypeptide chain forming a figure-of-eight knot, their binding to BV leads to shielding of many sites of proteolytic degradation due to additional stabilization of the entire protein structure. This makes NIR FPs with both cysteine residues in PAS and GAF domains less susceptible to cleavage by intracellular proteases.
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spelling pubmed-92670112022-07-09 Impact of Double Covalent Binding of BV in NIR FPs on Their Spectral and Physicochemical Properties Stepanenko, Olga V. Kuznetsova, Irina M. Turoverov, Konstantin K. Stepanenko, Olesya V. Int J Mol Sci Article Understanding the photophysical properties and stability of near-infrared fluorescent proteins (NIR FPs) based on bacterial phytochromes is of great importance for the design of efficient fluorescent probes for use in cells and in vivo. Previously, the natural ligand of NIR FPs biliverdin (BV) has been revealed to be capable of covalent binding to the inherent cysteine residue in the PAS domain (Cys15), and to the cysteine residue introduced into the GAF domain (Cys256), as well as simultaneously with these two residues. Here, based on the spectroscopic analysis of several NIR FPs with both cysteine residues in PAS and GAF domains, we show that the covalent binding of BV simultaneously with two domains is the reason for the higher quantum yield of BV fluorescence in these proteins as a result of rigid fixation of the chromophore in their chromophore-binding pocket. We demonstrate that since the attachment sites are located in different regions of the polypeptide chain forming a figure-of-eight knot, their binding to BV leads to shielding of many sites of proteolytic degradation due to additional stabilization of the entire protein structure. This makes NIR FPs with both cysteine residues in PAS and GAF domains less susceptible to cleavage by intracellular proteases. MDPI 2022-07-01 /pmc/articles/PMC9267011/ /pubmed/35806351 http://dx.doi.org/10.3390/ijms23137347 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Stepanenko, Olga V.
Kuznetsova, Irina M.
Turoverov, Konstantin K.
Stepanenko, Olesya V.
Impact of Double Covalent Binding of BV in NIR FPs on Their Spectral and Physicochemical Properties
title Impact of Double Covalent Binding of BV in NIR FPs on Their Spectral and Physicochemical Properties
title_full Impact of Double Covalent Binding of BV in NIR FPs on Their Spectral and Physicochemical Properties
title_fullStr Impact of Double Covalent Binding of BV in NIR FPs on Their Spectral and Physicochemical Properties
title_full_unstemmed Impact of Double Covalent Binding of BV in NIR FPs on Their Spectral and Physicochemical Properties
title_short Impact of Double Covalent Binding of BV in NIR FPs on Their Spectral and Physicochemical Properties
title_sort impact of double covalent binding of bv in nir fps on their spectral and physicochemical properties
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9267011/
https://www.ncbi.nlm.nih.gov/pubmed/35806351
http://dx.doi.org/10.3390/ijms23137347
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