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Fluorescence lifetime imaging microscopy (FLIM)-based Förster resonance energy transfer (FRET) measurement (FLIM-FRET) is one of the powerful methods for imaging of intracellular protein activities such as protein–protein interactions and conformational changes. Here, using saturation mutagenesis, we developed a dark yellow fluorescent protein named ShadowY that can serve as an acceptor for FLIM-FRET. ShadowY is spectrally similar to the previously reported dark YFP but has a much smaller quantum yield, greater extinction coefficient, and superior folding property. When ShadowY was paired with mEGFP or a Clover mutant (Clover T153M/F223R ) and applied to a single-molecule FRET sensor to monitor a light-dependent conformational change of the light-oxygen-voltage domain 2 (LOV2) in HeLa cells, we observed a large FRET signal change with low cell-to-cell variability, allowing for precise measurement of individual cell responses. In addition, an application of ShadowY to a separate-type Ras FRET sensor revealed an EGF-dependent large FRET signal increase. Thus, ShadowY in combination with mEGFP or Clover T153M/F223R  is a promising FLIM-FRET acceptor.

ShadowY: a dark yellow fluorescent protein for FLIM-based FRET measurement