ArticleScientific reports2015
Optimizing fluorescent protein trios for 3-Way FRET imaging of protein interactions in living cells.
Article in Scientific reports, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 15 papers.
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Who cites it
15 citing papers in PubMed.
- Characterization and structural basis for the brightness of mCLIFY: A novel monomeric and circularly permuted bright yellow fluorescent protein.Biophysical journal · 2025Article
- Characterization and structural basis for the brightness of mCLIFY: a novel monomeric and circularly permuted bright yellow fluorescent protein.Research square · 2024Article
- Article
- Specific detection of tau seeding activity in Alzheimer's disease using rationally designed biosensor cells.Molecular neurodegeneration · 2023Article
- Three-Fluorophore FRET Enables the Analysis of Ternary Protein Association in Living Plant Cells.Plants (Basel, Switzerland) · 2022Article
- Endosomes supporting fusion mediated by vesicular stomatitis virus glycoprotein have distinctive motion and acidification.Traffic (Copenhagen, Denmark) · 2022Article
- Flow cytometry based-FRET: basics, novel developments and future perspectives.Cellular and molecular life sciences : CMLS · 2022Review
- A Combined Acceptor Photobleaching and Donor Fluorescence Lifetime Imaging Microscopy Approach to Analyze Multi-Protein Interactions in Living Cells.Frontiers in molecular biosciences · 2021Article
- FRETting about the affinity of bimolecular protein-protein interactions.Protein science : a publication of the Protein Society · 2018Article
- Article
- Characterization of a spectrally diverse set of fluorescent proteins as FRET acceptors for mTurquoise2.Scientific reports · 2017Article
- A Guide to Fluorescent Protein FRET Pairs.Sensors (Basel, Switzerland) · 2016Review
- Article
- Positive Allosteric Modulation of Insect Olfactory Receptor Function by ORco Agonists.Frontiers in cellular neuroscience · 2016Article
- Three-Dimensional Reconstruction of Three-Way FRET Microscopy Improves Imaging of Multiple Protein-Protein Interactions.PloS one · 2016Article
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Authors and funding
2 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Powerful new methods have extended FRET microscopy to the imaging of three or more interacting proteins inside living cells. Here, we compared widely available fluorescent proteins to find the best trio for 3-Way FRET imaging. We focused on readily available cyan, yellow, and red proteins that have high quantum yields, large extinction coefficients and good photostability, which defined these candidate proteins: CyPet/mTFP1/mTurqoise2, mCitrine/YPet, and TagRFP/TagRFPt/mRuby2/mCherry. By taking advantage of the high structural similarity across the fluorescent proteins, we generated structurally similar, but photophysically distinct donor/acceptor and triple fluorophore fusion proteins and measured their FRET efficiencies inside living cells. Surprisingly, their published photophysical parameters and calculated Förster distances did not predict the best combinations of FPs. Using cycloheximide to inhibit protein synthesis, we found that the different FP maturation rates had a strong effect on the FRET efficiency. This effect was pronounced when comparing rapidly maturing yellow and slowly maturing red FPs. We found that red FPs with inferior photophysics gave superior FRET efficiencies because of faster maturation rates. Based on combined metrics for the FRET efficiency, fluorophore photophysics and fluorophore maturation we determined that Turqoise2, YPet and Cherry were the best available FPs for live cell 3-Way FRET measurements.
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