Abstract
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 (CloverT153M/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 CloverT153M/F223R is a promising FLIM-FRET acceptor.
Highlights
Protein conformational changes and protein–protein interactions form the basis of intracellular biochemical signal transduction
MEGFP–super REACh (sREACh) pair yields a superior Förster resonance energy transfer (FRET) signal, the spectral contamination of basal sREACh fluorescence owing to residual quantum efficiency (0.07) can produce unexpected artifacts, limiting applications of this pair[8]
We confirmed that the pairing of ShadowY with monomeric EGFP (mEGFP), Clover[13], or its mutant (CloverT153M/F223R) shows improved FRET signals with reduced cell-to-cell variability
Summary
Protein conformational changes and protein–protein interactions form the basis of intracellular biochemical signal transduction. As a pair of fluorescent proteins for FLIM-FRET, enhanced green fluorescent protein (EGFP) as an energy donor and red fluorescent protein (RFP) as an energy acceptor are frequently used[4, 5] Because this pair has well-separated emission spectra, this combination prevents spectral contamination due to the bleed-through of RFP fluorescence to the EGFP channel. FLIM-FRET requires only donor fluorescence (not acceptor fluorescence) for the detection of FRET5 By means of this feature, a fluorescent protein with a low quantum yield called resonance energy-accepting chromoprotein (REACh) was developed and applied to an acceptor of FRET7. MEGFP–sREACh pair yields a superior FRET signal, the spectral contamination of basal sREACh fluorescence owing to residual quantum efficiency (0.07) can produce unexpected artifacts, limiting applications of this pair[8] To overcome this limitation, a very dark fluorescent protein named ShadowG was developed by directed evolution using sREACh8. MEGFP–ShadowY and Clover mutant–ShadowY are good FLIM-FRET pairs
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