ShadowY: a dark yellow fluorescent protein for FLIM-based FRET measurement.
Sci Rep
; 7(1): 6791, 2017 07 28.
Article
em En
| MEDLINE
| ID: mdl-28754922
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Transferência Ressonante de Energia de Fluorescência
/
Proteínas de Fluorescência Verde
Limite:
Humans
Idioma:
En
Revista:
Sci Rep
Ano de publicação:
2017
Tipo de documento:
Article
País de afiliação:
Japão