Optimizing fluorescent protein trios for 3-Way FRET imaging of protein interactions in living cells.

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.