Abstract
Measurement of Förster resonance energy transfer by fluorescence lifetime imaging microscopy (FLIM-FRET) is a powerful method for visualization of intracellular signaling activities such as protein-protein interactions and conformational changes of proteins. Here, we developed a dark green fluorescent protein (ShadowG) that can serve as an acceptor for FLIM-FRET. ShadowG is spectrally similar to monomeric enhanced green fluorescent protein (mEGFP) and has a 120-fold smaller quantum yield. When FRET from mEGFP to ShadowG was measured using an mEGFP-ShadowG tandem construct with 2-photon FLIM-FRET, we observed a strong FRET signal with low cell-to-cell variability. Furthermore, ShadowG was applied to a single-molecule FRET sensor to monitor a conformational change of CaMKII and of the light oxygen voltage (LOV) domain in HeLa cells. These sensors showed reduced cell-to-cell variability of both the basal fluorescence lifetime and response signal. In contrast to mCherry- or dark-YFP-based sensors, our sensor allowed for precise measurement of individual cell responses. When ShadowG was applied to a separate-type Ras FRET sensor, it showed a greater response signal than did the mCherry-based sensor. Furthermore, Ras activation and translocation of its effector ERK2 into the nucleus could be observed simultaneously. Thus, ShadowG is a promising FLIM-FRET acceptor.
Highlights
Measurement of Förster resonance energy transfer by fluorescence lifetime imaging microscopy (FLIM-FRET) is a powerful method for visualization of intracellular signaling activities such as proteinprotein interactions and conformational changes of proteins
We aimed to develop a darker super REACh (sREACh) by error-prone PCR-based random mutagenesis
To screen the library for a dark sREACh mutant that has a high extinction coefficient but a low quantum yield, we first identified vividly colored colonies under day light, assuming that this characteristic is due to high absorption by the fluorescent protein in the bacteria
Summary
Measurement of Förster resonance energy transfer by fluorescence lifetime imaging microscopy (FLIM-FRET) is a powerful method for visualization of intracellular signaling activities such as proteinprotein interactions and conformational changes of proteins. ShadowG was applied to a single-molecule FRET sensor to monitor a conformational change of CaMKII and of the light oxygen voltage (LOV) domain in HeLa cells. These sensors showed reduced cell-to-cell variability of both the basal fluorescence lifetime and response signal. The frequently used pair of fluorescent proteins for FLIM-FRET is EGFP as an energy donor and monomeric red (mRFP) or cherry (mCherry) fluorescent protein as an energy acceptor[5,6,7]. When ShadowG was paired with mEGFP with the A206K mutation (in EGFP) and was applied to FRET sensors, we observed a robust FRET signal with reduced cell-to-cell variability of both the basal and response signal, most likely due to the superior folding and maturation properties of ShadowG
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