ShadowY: a dark yellow fluorescent protein for FLIM-based FRET measurement

Abstract Fluorescence lifetime imaging microscopy (FLIM)-based Förster resonance energy transfer (FRET) measurement (FLIM-FRET) is one of the powerful methods for imaging of intracellular protein activities such as protein–protein interactions and conformational changes. Here, using saturation mutag...

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Autores principales:	Hideji Murakoshi, Akihiro C. E. Shibata
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/1a287e0a1fa24cb8b4a1ba840a3bed3d
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spelling	oai:doaj.org-article:1a287e0a1fa24cb8b4a1ba840a3bed3d2021-12-02T11:52:17ZShadowY: a dark yellow fluorescent protein for FLIM-based FRET measurement10.1038/s41598-017-07002-42045-2322https://doaj.org/article/1a287e0a1fa24cb8b4a1ba840a3bed3d2017-07-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-07002-4https://doaj.org/toc/2045-2322Abstract Fluorescence lifetime imaging microscopy (FLIM)-based Förster resonance energy transfer (FRET) measurement (FLIM-FRET) is one of the powerful methods for imaging of intracellular protein activities such as protein–protein interactions and conformational changes. Here, using saturation mutagenesis, we developed a dark yellow fluorescent protein named ShadowY that can serve as an acceptor for FLIM-FRET. ShadowY is spectrally similar to the previously reported dark YFP but has a much smaller quantum yield, greater extinction coefficient, and superior folding property. When ShadowY was paired with mEGFP or a Clover mutant (CloverT153M/F223R) and applied to a single-molecule FRET sensor to monitor a light-dependent conformational change of the light-oxygen-voltage domain 2 (LOV2) in HeLa cells, we observed a large FRET signal change with low cell-to-cell variability, allowing for precise measurement of individual cell responses. In addition, an application of ShadowY to a separate-type Ras FRET sensor revealed an EGF-dependent large FRET signal increase. Thus, ShadowY in combination with mEGFP or CloverT153M/F223R is a promising FLIM-FRET acceptor.Hideji MurakoshiAkihiro C. E. ShibataNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-10 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Hideji Murakoshi Akihiro C. E. Shibata ShadowY: a dark yellow fluorescent protein for FLIM-based FRET measurement
description	Abstract Fluorescence lifetime imaging microscopy (FLIM)-based Förster resonance energy transfer (FRET) measurement (FLIM-FRET) is one of the powerful methods for imaging of intracellular protein activities such as protein–protein interactions and conformational changes. Here, using saturation mutagenesis, we developed a dark yellow fluorescent protein named ShadowY that can serve as an acceptor for FLIM-FRET. ShadowY is spectrally similar to the previously reported dark YFP but has a much smaller quantum yield, greater extinction coefficient, and superior folding property. When ShadowY was paired with mEGFP or a Clover mutant (CloverT153M/F223R) and applied to a single-molecule FRET sensor to monitor a light-dependent conformational change of the light-oxygen-voltage domain 2 (LOV2) in HeLa cells, we observed a large FRET signal change with low cell-to-cell variability, allowing for precise measurement of individual cell responses. In addition, an application of ShadowY to a separate-type Ras FRET sensor revealed an EGF-dependent large FRET signal increase. Thus, ShadowY in combination with mEGFP or CloverT153M/F223R is a promising FLIM-FRET acceptor.
format	article
author	Hideji Murakoshi Akihiro C. E. Shibata
author_facet	Hideji Murakoshi Akihiro C. E. Shibata
author_sort	Hideji Murakoshi
title	ShadowY: a dark yellow fluorescent protein for FLIM-based FRET measurement
title_short	ShadowY: a dark yellow fluorescent protein for FLIM-based FRET measurement
title_full	ShadowY: a dark yellow fluorescent protein for FLIM-based FRET measurement
title_fullStr	ShadowY: a dark yellow fluorescent protein for FLIM-based FRET measurement
title_full_unstemmed	ShadowY: a dark yellow fluorescent protein for FLIM-based FRET measurement
title_sort	shadowy: a dark yellow fluorescent protein for flim-based fret measurement
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/1a287e0a1fa24cb8b4a1ba840a3bed3d
work_keys_str_mv	AT hidejimurakoshi shadowyadarkyellowfluorescentproteinforflimbasedfretmeasurement AT akihiroceshibata shadowyadarkyellowfluorescentproteinforflimbasedfretmeasurement
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ShadowY: a dark yellow fluorescent protein for FLIM-based FRET measurement

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