An improved cyan fluorescent protein variant useful for FRET

Rizzo, Mark A.; Springer, Gerald H.; Granada, Butch; Piston, David W.

doi:10.1038/nbt945

Cited by 1,000 publications

(915 citation statements)

References 20 publications

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“…To our knowledge, the fluorescent protein variants we generated, in a wild-type GFP backbone, are not commercially available. The GFP and BFP sequences are similar those previously reported (Zernicka-Goetz et al, 1996), and the final CFP we used is similar to a variant called cerulean (Rizzo et al, 2004). An internal EcoRI restriction site present in wild-type DsRed was removed by overlap PCR without altering any codons.…”

Section: Site-directed Mutagenesis and Codon Optimizationsupporting

confidence: 56%

Optimized green fluorescent protein variants provide improved single cell resolution of transgene expression in ascidian embryos

Zeller

Weldon

Pellatiro

et al. 2005

Developmental Dynamics

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The green fluorescent protein (GFP) is used extensively to monitor gene expression and protein localization in living cells, particularly in developing embryos from a variety of species. Several GFP mutations have been characterized that improve protein expression and alter the emission spectra to produce proteins that emit green, blue, cyan, and yellow wavelengths. DsRed and its variants encode proteins that emit in the orange to red wavelengths. Many of these commercially available fluorescent proteins have been "codon optimized" for maximal levels of expression in mammalian cells. We have generated several fluorescent protein color variants that have been codon optimized for maximal expression in the ascidian Ciona intestinalis. By analyzing quantitative time-lapse recordings of transgenic embryos, we demonstrate that, in general, our Ciona optimized variants are detected and expressed at higher levels than commercially available fluorescent proteins. We show that three of these proteins, expressed simultaneously in different spatial domains within the same transgenic embryo are easily detectable using optimized fluorescent filter sets for epifluorescent microscopy. Coupled with recently developed quantitative imaging techniques, our GFP variants should provide useful reagents for monitoring the simultaneous expression of multiple genes in transgenic ascidian embryos. Developmental Dynamics 235:456 -467, 2006.

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Section: Site-directed Mutagenesis and Codon Optimizationsupporting

confidence: 56%

Optimized green fluorescent protein variants provide improved single cell resolution of transgene expression in ascidian embryos

Zeller

Weldon

Pellatiro

et al. 2005

Developmental Dynamics

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“…One shorter lifetime of 1.1 ns contributing for 33% and one longer lifetime of 3.7 ns contributing for 67% could be recovered. The departure of single-exponential fluorescence decay is also observed by others [18][19][20]. The presence of multiple fluorescent states is most probably due to two different conformations of ECFP in the crystal structure in which two amino acids, Tyr145 and His148, are positioned differently with respect to the fluorophore [21].…”

Section: Fluorescence Decays In Different Water-glycerol Mixturesmentioning

confidence: 70%

“…The interconversion between both conformations is slow on the fluorescence time scale. From site-directed mutagenesis studies, His148 has been found to be the major quencher of the fluorophore [19].…”

Section: Fluorescence Decays In Different Water-glycerol Mixturesmentioning

confidence: 99%

Effects of Refractive Index and Viscosity on Fluorescence and Anisotropy Decays of Enhanced Cyan and Yellow Fluorescent Proteins

et al. 2005

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The fluorescence lifetime strongly depends on the immediate environment of the fluorophore. Timeresolved fluorescence measurements of the enhanced forms of ECFP and EYFP in water-glycerol mixtures were performed to quantify the effects of the refractive index and viscosity on the fluorescence lifetimes of these proteins. The experimental data show for ECFP and EYFP two fluorescence lifetime components: one short lifetime of about 1 ns and a longer lifetime of about 3.7 ns of ECFP and for EYFP 3.4. The fluorescence of ECFP is very heterogeneous, which can be explained by the presence of two populations: a conformation (67% present) where the fluorophore is less quenched than in the other conformation (33% present). The fluorescence decay of EYFP is much more homogeneous and the amplitude of the short fluorescence lifetime is about 5%. The fluorescence anisotropy decays show that the rotational correlation time of both proteins scales with increasing viscosity of the solvent similarly as shown earlier for GFP. The rotational correlation times are identical for ECFP and EYFP, which can be expected since both proteins have the same shape and size. The only difference observed is the slightly lower initial anisotropy for ECFP as compared to the one of EYFP.

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“…In order to get more consistent energy transfer rates, donor species with monoexponential fluorescence decay kinetics should replace ECFP. Candidates may be cerulean with a monoexponential decay paired with yellow fluorescent protein (30), GFP and red fluorescent protein variants (31,32) or YFP paired with mRFP (33). Presently, the fluorescence lifetime s 2 , the effective lifetime s eff , as well as the ratio of amplitudes a 1 /a 2 (describing the contributions of short-lived and long-lived fluorescence) appear to be appropriate parameters for measuring nonradiative energy transfer.…”

Section: Discussionmentioning

confidence: 99%

A membrane‐bound FRET‐based caspase sensor for detection of apoptosis using fluorescence lifetime and total internal reflection microscopy

et al. 2008

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A caspase sensor based on Förster resonance energy transfer between fluorescent proteins is reported. Enhanced cyan fluorescent protein anchored to the inner leaflet of the plasma membrane of living cells is optically excited by an evanescent electromagnetic field and transfers its excitation energy via a spacer (DEVD) to an enhanced yellow fluorescent protein. Upon apoptosis, DEVD is cleaved and energy transfer is disrupted, as proven by pronounced changes in fluorescence spectra and decay times. Fluorescence spectroscopy and lifetime imaging (FLIM) is combined with total internal reflection fluorescence microscopy (TIRFM) for selective detection of this membrane-bound caspase sensor. Fluorophores of the cytoplasm are thus excluded, and the signal-to-background ratio is increased considerably. In comparison with conventional or laser scanning microscopy, this permits long-term observation of apoptosis in live cell cultures using very low absorption and avoiding light-induced damages of the samples. ' 2008International Society for Advancement of Cytometry Key terms caspase sensor; apoptosis; Förster resonance energy transfer; fluorescence lifetime microscopy; total internal reflection microscopy; live cell microscopy THE measurement of caspase activities is commonly used to detect and investigate programmed cell death. Cyan fluorescent protein (CFP) fused to yellow fluorescent protein (YFP) by a caspase-sensitive amino acid peptide (DEVD) has been developed as an indicator of caspase-3 activity in living cells (1,2). The peptide linker between CFP and YFP is short enough to bring the two fluorescent proteins in close proximity to each other resulting in Förster resonance fluorescence energy transfer [FRET; (3)]. Upon induction of apoptosis, FRET is interrupted due to cleavage of the linker by caspase-3, and thus loss of FRET is a direct indicator of caspase activity. The sensitivity of this genetically encoded caspase sensor has been improved by optimizing the amino acid sequence flanking DEVD for higher FRET efficiency and thus improving the dynamic range of signal generation (4,5). Caspase sensor constructs following this principle have since been used to investigate caspase activity in living cells. For example, the rate of apoptosis of whole cell populations was determined (6-8), or the temporally distinct onset of apoptosis in single cells within whole cell collectives was detected (8). Furthermore, cleavage sequences specific for different caspases were used to reveal the activation of these caspases in the same cell (9,10).Detection of events of such dynamic occurrence often requires a temporal and spatial resolution in data acquisition that are only obtainable by real-time or timelapse recordings. So far, changes in FRET of caspase sensor probes have been mostly detected by calculating the ratio of acceptor (YFP) and donor (CFP) emission using conventional standard fluorescence or confocal laser scanning microscopy (4,11). However, both microscopic methods can be harmful to living cells because...

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An improved cyan fluorescent protein variant useful for FRET

Cited by 1,000 publications

References 20 publications

Optimized green fluorescent protein variants provide improved single cell resolution of transgene expression in ascidian embryos

Optimized green fluorescent protein variants provide improved single cell resolution of transgene expression in ascidian embryos

Effects of Refractive Index and Viscosity on Fluorescence and Anisotropy Decays of Enhanced Cyan and Yellow Fluorescent Proteins

A membrane‐bound FRET‐based caspase sensor for detection of apoptosis using fluorescence lifetime and total internal reflection microscopy

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