A dark green fluorescent protein as an acceptor for measurement of Förster resonance energy transfer
2015
Details
- Autor(en) / Beteiligte
- Titel
- A dark green fluorescent protein as an acceptor for measurement of Förster resonance energy transfer
- Ist Teil von
- Scientific reports, 2015-10, Vol.5 (1), p.15334, Article 15334
- Ort / Verlag
- England: Nature Publishing Group
- Erscheinungsjahr
- 2015
- Link zum Volltext
- Quelle
- Free E-Journal (出版社公開部分のみ）
- Beschreibungen/Notizen
- Measurement of Förster resonance energy transfer by fluorescence lifetime imaging microscopy (FLIM-FRET) is a powerful method for visualization of intracellular signaling activities such as protein-protein interactions and conformational changes of proteins. Here, we developed a dark green fluorescent protein (ShadowG) that can serve as an acceptor for FLIM-FRET. ShadowG is spectrally similar to monomeric enhanced green fluorescent protein (mEGFP) and has a 120-fold smaller quantum yield. When FRET from mEGFP to ShadowG was measured using an mEGFP-ShadowG tandem construct with 2-photon FLIM-FRET, we observed a strong FRET signal with low cell-to-cell variability. Furthermore, ShadowG was applied to a single-molecule FRET sensor to monitor a conformational change of CaMKII and of the light oxygen voltage (LOV) domain in HeLa cells. These sensors showed reduced cell-to-cell variability of both the basal fluorescence lifetime and response signal. In contrast to mCherry- or dark-YFP-based sensors, our sensor allowed for precise measurement of individual cell responses. When ShadowG was applied to a separate-type Ras FRET sensor, it showed a greater response signal than did the mCherry-based sensor. Furthermore, Ras activation and translocation of its effector ERK2 into the nucleus could be observed simultaneously. Thus, ShadowG is a promising FLIM-FRET acceptor.
- Sprache
- Englisch
- Identifikatoren
- ISSN: 2045-2322eISSN: 2045-2322DOI: 10.1038/srep15334Titel-ID: cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_4606784
- Format
- –
- Schlagworte
- Bacterial Proteins - chemistry, Bacterial Proteins - genetics, Bacterial Proteins - metabolism, Ca2+/calmodulin-dependent protein kinase II, Calcium-Calmodulin-Dependent Protein Kinase Type 2 - chemistry, Calcium-Calmodulin-Dependent Protein Kinase Type 2 - genetics, Calcium-Calmodulin-Dependent Protein Kinase Type 2 - metabolism, Fluorescence Resonance Energy Transfer, Green fluorescent protein, Green Fluorescent Proteins - chemistry, Green Fluorescent Proteins - genetics, Green Fluorescent Proteins - metabolism, HEK293 Cells, HeLa Cells, Humans, Intracellular signalling, Luminescent Proteins - chemistry, Luminescent Proteins - genetics, Luminescent Proteins - metabolism, Microscopy, Fluorescence, Mutagenesis, Plasmids - metabolism, Protein folding, Protein interaction, Protein Interaction Domains and Motifs, Proteins, Quantum Theory, ras Proteins - chemistry, ras Proteins - genetics, ras Proteins - metabolism, Recombinant Fusion Proteins - biosynthesis, Recombinant Fusion Proteins - chemistry, Recombinant Fusion Proteins - genetics, Red Fluorescent Protein, Sensors