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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...
Autores principales: | , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The Royal Society of Chemistry
2022
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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. |
format | Online Article Text |
id | pubmed-9535469 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | The Royal Society of Chemistry |
record_format | MEDLINE/PubMed |
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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