title: A dark green fluorescent protein as an acceptor for measurement of Förster resonance energy transfer.
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.
authors.affiliation: Supportive Center for Brain Research, National Institute for Physiological Sciences, Okazaki, Aichi 444-8585, Japan.|Department of Physiological Sciences, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Aichi 444-8585, Japan.|Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0012, Japan.
authors.forename: Hideji
authors.initials: H
authors.lastname: Murakoshi
authors.affiliation: Supportive Center for Brain Research, National Institute for Physiological Sciences, Okazaki, Aichi 444-8585, Japan.
authors.forename: Akihiro C E
authors.initials: ACE
authors.lastname: Shibata
authors.affiliation: Division of Homeostatic Development, National Institute for Physiological Sciences, Okazaki, Aichi 444-8585, Japan.
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Nakahata
authors.affiliation: Department of Physiological Sciences, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Aichi 444-8585, Japan.|Division of Homeostatic Development, National Institute for Physiological Sciences, Okazaki, Aichi 444-8585, Japan.|Core Research for Evolutional Science and Technology, Japan Science and Technology Agency (JST), Kawaguchi, Saitama 332-0012, Japan.
authors.forename: Junichi
authors.initials: J
authors.lastname: Nabekura
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D016296:Mutagenesis; D010957:Plasmids / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D011789:Quantum Theory; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D018631:ras Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D001426:Bacterial Proteins; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D018631:ras Proteins; D000097573:Red Fluorescent Protein; C485184:enhanced green fluorescent protein; C054562:yellow fluorescent protein, Bacteria