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Fluorescence resonance energy transfer of GFP and YFP by spectral imaging and quantitative acceptor photobleaching
Author(s) -
DINANT C.,
VAN ROYEN M. E.,
VERMEULEN W.,
HOUTSMULLER A. B.
Publication year - 2008
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.1365-2818.2008.02020.x
Subject(s) - förster resonance energy transfer , photobleaching , yellow fluorescent protein , green fluorescent protein , fluorescence microscope , fluorescence , fluorophore , chemistry , biophysics , fluorescence lifetime imaging microscopy , biology , optics , biochemistry , physics , gene
Summary To study protein–protein interactions by fluorescence energy transfer (FRET), the proteins of interest are tagged with either a donor or an acceptor fluorophore. For efficient FRET, fluorophores need to have a reasonable overlap of donor emission and acceptor excitation spectra. However, given the relatively small Stokes shift of conventional fluorescent proteins, donor and acceptor pairs with high FRET efficiencies have emission spectra that are difficult to separate. GFP and YFP are widely used in fluorescence microscopy studies. The spectral qualities of GFP and YFP make them one of the most efficient FRET donor–acceptor couples available. However, the emission peaks of GFP (510 nm) and YFP (527 nm) are spectrally too close for separation by conventional fluorescence microscopy. Difficulties in simultaneous detection of GFP and YFP with a fluorescence microscope are eliminated when spectral imaging and subsequent linear unmixing are applied. This allows FRET microscopy using these tags to study protein–protein interactions. We adapted the linear unmixing procedure from commercially available software (Zeiss) for use with acceptor photobleaching FRET using GFP and YFP as FRET pair. FRET efficiencies up to 52% for a GFP‐YFP fusion protein were measured. To investigate the applicability of the procedure, we used two constituents of the nucleotide excision repair system, which removes UV‐induced single‐strand DNA damage. ERCC1 and XPF form a heterodimeric 5′ endonuclease in nucleotide excision repair. FRET between ERCC1‐GFP and XPF‐YFP occurs with an efficiency of 30%.