Practical and reliable FRET/FLIM pair of fluorescent proteins

Abstract

Background: In spite of a great number of monomeric fluorescent proteins developed in the recent years, the reported fluorescent protein-based FRET pairs are still characterized by a number of disadvantageous features, complicating their use as reporters in cell biology and for high-throughput cell-based screenings.

Results: Here we screened some of the recently developed monomeric protein pairs to find the optimal combination, which would provide high dynamic range FRET changes, along with high pH- and photo-stability, fast maturation and bright fluorescence, and reliable detection in any fluorescent imaging system. Among generated FRET pairs, we have selected TagGFP-TagRFP, combining all the mentioned desirable characteristics. On the basis of this highly efficient FRET pair, we have generated a bright, high contrast, pH- and photo-stable apoptosis reporter, named CaspeR3 (Caspase 3 Reporter).

Conclusion: The combined advantages suggest that the TagGFP-TagRFP is one of the most efficient green/red couples available to date for FRET/FLIM analyses to monitor interaction of proteins of interest in living cells and to generate FRET-based sensors for various applications. CaspeR3 provides reliable detection of apoptosis, and should become a popular tool both for cell biology studies and high throughput screening assays.

Figure 1

Spectral response and caspase sensing of CaspeR3. A. Excitation (dashed lines) and emission (solid lines) spectra for TagGFP (green) and TagRFP (red). Spectral overlap is filled with gray. B. Emission spectra of CaspeR3 before (dashed line) and after digestion by Caspase 3 (solid line). C. Green-to-red emission ratio change of CaspeR3 upon staurosporine-induced apoptosis. Approximately 40–50 min after staurosporine infusion, cells demonstrated pronounced changes fluorescence signal ratio. Emission ratio shown for 5 cells, time point aligned to the median of ratio changes, individual for each cell. Excitation at 488 nm, emission was detected at 500–530 nm and 560–600 nm. D. TagGFP fluorescence lifetime τ_φ (solid lines) and τ_M (dashed lines) changes for CaspeR3 during staurosporine-induced apoptosis. Excitation was at 488 nm and donor fluorescence emission was passed through a 500–530 nm bandpass filter. E. Two channel fluorescence imaging of CaspeR3 upon staurosporine-induced apoptosis in HeLa cells. Time, in minutes, is shown after staurosporine infusion.