A new pair for inter- and intra-molecular FRET measurement
- Institute of Biophysics and Biochemistry, School of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074 (China)
- Institute of Biophysics and Biochemistry, School of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074 (China) and Institute of Biophysics and Biochemistry, School of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074 (China)
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 Ca{sup 2+}-sensor cameleon with EGFP/TDimer2, dynamic changes in cytosolic free Ca{sup 2+} concentrations were observed with 488 nm 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.
- OSTI ID:
- 20709189
- Journal Information:
- Biochemical and Biophysical Research Communications, Journal Name: Biochemical and Biophysical Research Communications Journal Issue: 3 Vol. 330; ISSN 0006-291X; ISSN BBRCA9
- Country of Publication:
- United States
- Language:
- English
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