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Spatial Structure of NanoFAST in the Apo State and in Complex with its Fluorogen HBR-DOM2

NanoFAST is a fluorogen-activating protein and can be considered one of the smallest encodable fluorescent tags. Being a shortened variant of another fluorescent tag, FAST, nanoFAST works nicely only with one out of all known FAST ligands. This substantially limits the applicability of this protein....

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Autores principales: Lushpa, Vladislav A., Baleeva, Nadezhda S., Goncharuk, Sergey A., Goncharuk, Marina V., Arseniev, Alexander S., Baranov, Mikhail S., Mineev, Konstantin S.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9570328/
https://www.ncbi.nlm.nih.gov/pubmed/36232662
http://dx.doi.org/10.3390/ijms231911361
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author Lushpa, Vladislav A.
Baleeva, Nadezhda S.
Goncharuk, Sergey A.
Goncharuk, Marina V.
Arseniev, Alexander S.
Baranov, Mikhail S.
Mineev, Konstantin S.
author_facet Lushpa, Vladislav A.
Baleeva, Nadezhda S.
Goncharuk, Sergey A.
Goncharuk, Marina V.
Arseniev, Alexander S.
Baranov, Mikhail S.
Mineev, Konstantin S.
author_sort Lushpa, Vladislav A.
collection PubMed
description NanoFAST is a fluorogen-activating protein and can be considered one of the smallest encodable fluorescent tags. Being a shortened variant of another fluorescent tag, FAST, nanoFAST works nicely only with one out of all known FAST ligands. This substantially limits the applicability of this protein. To find the reason for such a behavior, we investigated the spatial structure and dynamics of nanoFAST, both in the apo state and in the complex with its fluorogen molecule, using the solution NMR spectroscopy. We showed that the truncation of FAST did not affect the structure of the remaining part of the protein. Our data suggest that the deleted N-terminus of FAST destabilizes the C-terminal domain in the apo state. While it does not contact the fluorogen directly, it serves as a free energy reservoir that enhances the ligand binding propensity of the protein. The structure of nanoFAST/HBR-DOM2 complex reveals the atomistic details of nanoFAST interactions with the rhodanine-based ligands and explains the ligand specificity. NanoFAST selects ligands with the lowest dissociation constants, 2,5-disubstituted 4-hydroxybenzyldienerhodainines, which allow the non-canonical intermolecular CH–N hydrogen bonding and provide the optimal packing of the ligand within the hydrophobic cavity of the protein.
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spelling pubmed-95703282022-10-17 Spatial Structure of NanoFAST in the Apo State and in Complex with its Fluorogen HBR-DOM2 Lushpa, Vladislav A. Baleeva, Nadezhda S. Goncharuk, Sergey A. Goncharuk, Marina V. Arseniev, Alexander S. Baranov, Mikhail S. Mineev, Konstantin S. Int J Mol Sci Article NanoFAST is a fluorogen-activating protein and can be considered one of the smallest encodable fluorescent tags. Being a shortened variant of another fluorescent tag, FAST, nanoFAST works nicely only with one out of all known FAST ligands. This substantially limits the applicability of this protein. To find the reason for such a behavior, we investigated the spatial structure and dynamics of nanoFAST, both in the apo state and in the complex with its fluorogen molecule, using the solution NMR spectroscopy. We showed that the truncation of FAST did not affect the structure of the remaining part of the protein. Our data suggest that the deleted N-terminus of FAST destabilizes the C-terminal domain in the apo state. While it does not contact the fluorogen directly, it serves as a free energy reservoir that enhances the ligand binding propensity of the protein. The structure of nanoFAST/HBR-DOM2 complex reveals the atomistic details of nanoFAST interactions with the rhodanine-based ligands and explains the ligand specificity. NanoFAST selects ligands with the lowest dissociation constants, 2,5-disubstituted 4-hydroxybenzyldienerhodainines, which allow the non-canonical intermolecular CH–N hydrogen bonding and provide the optimal packing of the ligand within the hydrophobic cavity of the protein. MDPI 2022-09-26 /pmc/articles/PMC9570328/ /pubmed/36232662 http://dx.doi.org/10.3390/ijms231911361 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
Lushpa, Vladislav A.
Baleeva, Nadezhda S.
Goncharuk, Sergey A.
Goncharuk, Marina V.
Arseniev, Alexander S.
Baranov, Mikhail S.
Mineev, Konstantin S.
Spatial Structure of NanoFAST in the Apo State and in Complex with its Fluorogen HBR-DOM2
title Spatial Structure of NanoFAST in the Apo State and in Complex with its Fluorogen HBR-DOM2
title_full Spatial Structure of NanoFAST in the Apo State and in Complex with its Fluorogen HBR-DOM2
title_fullStr Spatial Structure of NanoFAST in the Apo State and in Complex with its Fluorogen HBR-DOM2
title_full_unstemmed Spatial Structure of NanoFAST in the Apo State and in Complex with its Fluorogen HBR-DOM2
title_short Spatial Structure of NanoFAST in the Apo State and in Complex with its Fluorogen HBR-DOM2
title_sort spatial structure of nanofast in the apo state and in complex with its fluorogen hbr-dom2
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9570328/
https://www.ncbi.nlm.nih.gov/pubmed/36232662
http://dx.doi.org/10.3390/ijms231911361
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