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[25] Homo-FRET versus hetero-FRET to probe homodimers in living cells

2003; Academic Press; Linguagem: Inglês

10.1016/s0076-6879(03)60129-1

1557-7988

Marc Tramier, Tristan Piolot, Isabelle Gautier, Vincent Mignotte, J. Coppey, Klaus Kemnitz, Christiane Durieux, Maïté Coppey‐Moisan,

Photoreceptor and optogenetics research

Publisher Summary The purpose of this chapter is to provide information on the homo-fluorescence resonance energy transfer (FRET) versus hetero-FRET to probe homodimers in living cells. FRET is a nonradiative phenomenon in which energy is transferred from a donor fluorophore to an acceptor chromophore with an efficiency that depends on the distance between the two chromophores, the extent of overlap between the donor emission and acceptor excitation spectra, the quantum yield of the donor, and the relative orientation of the donor and acceptor. For homo-FRET, because the photophysical properties of the two donor molecules are the same, the excitation energy is reversibly transferred between the fluorescent tags. Time-resolved fluorescence anisotropy monitors any process that changes the polarization of the emitted fluorescence during the excited state. Consequently, the fluorescence anisotropy decay depends on (1) rotational movements of the fluorescent molecules and (2) energy transfer taking place within the fluorescence time scale. In addition, according to the type of interaction, hetero-or homodimer, the methodology, hetero- or homo-FRET, must be judiciously chosen to obtain the best information about structural data within the macromolecular complex.