A new pair for inter- and intra-molecular FRET measurement.

Fluorescence resonance energy transfer between mutant green fluorescent proteins provides powerful means to monitor in vivo protein-protein proximity and intracellular signaling. However, the current widely applied FRET pair of this class (CFP/YFP) requires excitation by expensive UV lasers, thereby hindering FRET imaging on many confocal microscopes. Further challenges arise from the large spectral overlap of CFP/YFP emission. Another FRET pair GFP/DsRed could obviate such limitations. However, the use of DsRed as a FRET acceptor is hampered by several critical problems, including a slow and incomplete maturation and obligate tetramerization. A tandem dimer mutant of DsRed (TDimer2) has similar spectral properties as those of DsRed. The rapid maturation and non-oligomerization make TDimer2 a promising substitute for DsRed in FRET experiments. Here, we have explored the possibility of using TDimer2 as a FRET acceptor for the donor EGFP. FRET was demonstrated between the EGFP-TDimer2 chimeric fusion protein. By substituting CFP/YFP in the Ca2+-sensor cameleon with EGFP/TDimer2, dynamic changes in cytosolic free Ca2+ concentrations were observed with 488nm excitation under conventional wide-field microscopy. The EGFP/TDimer2 pair was further successfully employed to monitor inter-molecular interaction between Syntaxin and SNAP25. These results reveal EGFP/TDimer2 as a promising FRET pair in monitoring intra-molecular conformation change as well as inter-molecular interaction.