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A disulphide bond-mediated hetero-dimer of a hemoprotein and a fluorescent protein exhibiting efficient energy transfer

Artificial protein hetero-dimerization is one of the promising strategies to construct protein-based chemical tools. In this work, cytochrome b(562), an electron transfer hemoprotein, and green fluorescent protein (GFP) mutants with cysteine residues added to their surfaces were conjugated via a pyr...

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Detalles Bibliográficos
Autores principales: Soon, Julian Wong, Oohora, Koji, Hayashi, Takashi
Formato: Online Artículo Texto
Lenguaje:English
Publicado: The Royal Society of Chemistry 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9535469/
https://www.ncbi.nlm.nih.gov/pubmed/36320522
http://dx.doi.org/10.1039/d2ra05249k
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author Soon, Julian Wong
Oohora, Koji
Hayashi, Takashi
author_facet Soon, Julian Wong
Oohora, Koji
Hayashi, Takashi
author_sort Soon, Julian Wong
collection PubMed
description Artificial protein hetero-dimerization is one of the promising strategies to construct protein-based chemical tools. In this work, cytochrome b(562), an electron transfer hemoprotein, and green fluorescent protein (GFP) mutants with cysteine residues added to their surfaces were conjugated via a pyridyl disulphide-based thiol–disulfide exchange reaction. The eight hetero-dimers, which have cysteine residues at different positions to form the disulphide bonds, were obtained and characterized by gel-electrophoresis, mass spectrometry and size exclusion chromatography. The fluorescence properties of the hetero-dimers were evaluated by fluorescence spectroscopy and fluorescence lifetime measurements. Efficient photoinduced energy transfer from the GFP chromophore to the heme cofactor was observed in each of the hetero-dimers. The energy transfer efficiency is strongly dependent on the cross-linking residues, reaching 96%. Furthermore, the estimated Förster distance and the structure-based maximum possible distances of the donor and acceptor suggest that one of the hetero-dimers has a rigid protein–protein structure with favourable properties for energy transfer. The disulphide bond-mediated protein hetero-dimerization is useful for screening functional protein systems towards further developments.
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spelling pubmed-95354692022-10-31 A disulphide bond-mediated hetero-dimer of a hemoprotein and a fluorescent protein exhibiting efficient energy transfer Soon, Julian Wong Oohora, Koji Hayashi, Takashi RSC Adv Chemistry Artificial protein hetero-dimerization is one of the promising strategies to construct protein-based chemical tools. In this work, cytochrome b(562), an electron transfer hemoprotein, and green fluorescent protein (GFP) mutants with cysteine residues added to their surfaces were conjugated via a pyridyl disulphide-based thiol–disulfide exchange reaction. The eight hetero-dimers, which have cysteine residues at different positions to form the disulphide bonds, were obtained and characterized by gel-electrophoresis, mass spectrometry and size exclusion chromatography. The fluorescence properties of the hetero-dimers were evaluated by fluorescence spectroscopy and fluorescence lifetime measurements. Efficient photoinduced energy transfer from the GFP chromophore to the heme cofactor was observed in each of the hetero-dimers. The energy transfer efficiency is strongly dependent on the cross-linking residues, reaching 96%. Furthermore, the estimated Förster distance and the structure-based maximum possible distances of the donor and acceptor suggest that one of the hetero-dimers has a rigid protein–protein structure with favourable properties for energy transfer. The disulphide bond-mediated protein hetero-dimerization is useful for screening functional protein systems towards further developments. The Royal Society of Chemistry 2022-10-06 /pmc/articles/PMC9535469/ /pubmed/36320522 http://dx.doi.org/10.1039/d2ra05249k Text en This journal is © The Royal Society of Chemistry https://creativecommons.org/licenses/by-nc/3.0/
spellingShingle Chemistry
Soon, Julian Wong
Oohora, Koji
Hayashi, Takashi
A disulphide bond-mediated hetero-dimer of a hemoprotein and a fluorescent protein exhibiting efficient energy transfer
title A disulphide bond-mediated hetero-dimer of a hemoprotein and a fluorescent protein exhibiting efficient energy transfer
title_full A disulphide bond-mediated hetero-dimer of a hemoprotein and a fluorescent protein exhibiting efficient energy transfer
title_fullStr A disulphide bond-mediated hetero-dimer of a hemoprotein and a fluorescent protein exhibiting efficient energy transfer
title_full_unstemmed A disulphide bond-mediated hetero-dimer of a hemoprotein and a fluorescent protein exhibiting efficient energy transfer
title_short A disulphide bond-mediated hetero-dimer of a hemoprotein and a fluorescent protein exhibiting efficient energy transfer
title_sort disulphide bond-mediated hetero-dimer of a hemoprotein and a fluorescent protein exhibiting efficient energy transfer
topic Chemistry
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9535469/
https://www.ncbi.nlm.nih.gov/pubmed/36320522
http://dx.doi.org/10.1039/d2ra05249k
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