title: A new pair for inter- and intra-molecular FRET measurement.
abstract: 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.
authors.affiliation: Institute of Biophysics and Biochemistry, School of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, PR China.
authors.forename: Xiaofei
authors.initials: X
authors.lastname: Yang
authors.forename: Pingyong
authors.initials: P
authors.lastname: Xu
authors.forename: Tao
authors.initials: T
authors.lastname: Xu
mesh_terms: D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism; D038761:Photobleaching; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D022622:Shiga Toxin 1 / Q000235:genetics / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25; D013997:Time Factors
chemical_list: D008164:Luminescent Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D022622:Shiga Toxin 1; C494210:Snap25 protein, rat; D050825:Synaptosomal-Associated Protein 25; C413663:fluorescent protein 583; D049452:Green Fluorescent Proteins; D002118:Calcium