Fluorescence resonance energy transfer (FRET) between mutant green fluorescent proteins (GFP) provides powerful means to monitor in vivo protein-protein proximity and intracellular messengers. However, the leading FRET pair of this class (CFP/YFP) entails suboptimal donor excitation by Argon lasers, thereby hindering FRET imaging on many confocal microscopes. Further challenges arise from the large spectral overlap of CFP/YFP emission. By contrast, DsRed, along with other members of a growing family of red-shifted sea coral fluorophores, features spectra that could obviate such limitations, using DsRed as FRET acceptor, and GFP or CFP as donor. Nonetheless, DsRed suffers from slow chromophore maturation, which confounds quantitative FRET. Here, we develop strategies minimizing the resulting complexity: 1), Pulsed activation of inducible promoters, driving expression of DsRed-tagged molecules, yields a uniform bolus of mature fluorophore; 2), The 33-FRET detection algorithm, adapted for CFP/DsRed and GFP/DsRed, proves insensitive to distortion by slow maturation. We thus show that DsRed supports strong FRET in CFP-DsRed or GFP-DsRed concatemers. These results reveal the promise of sea coral fluorophores like DsRed as FRET partners with GFP or CFP.
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