A dark green fluorescent protein as an acceptor for measurement of Förster resonance energy transfer

Sci Rep. 2015 Oct 15;5:15334. doi: 10.1038/srep15334.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2 / chemistry
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2 / genetics
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2 / metabolism
  • Fluorescence Resonance Energy Transfer*
  • Green Fluorescent Proteins / chemistry
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Luminescent Proteins / chemistry
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism*
  • Microscopy, Fluorescence
  • Mutagenesis
  • Plasmids / metabolism
  • Protein Interaction Domains and Motifs
  • Quantum Theory
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • ras Proteins / chemistry
  • ras Proteins / genetics
  • ras Proteins / metabolism


  • Bacterial Proteins
  • Luminescent Proteins
  • Recombinant Fusion Proteins
  • enhanced green fluorescent protein
  • red fluorescent protein
  • yellow fluorescent protein, Bacteria
  • Green Fluorescent Proteins
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2
  • ras Proteins