Circular dichroism spectroscopy of fluorescent proteins

Visser, Nina V.; Hink, Mark A.; Borst, Jan Willem; Krogt, G.N.M. van der; Visser, Antonie J. W. G.

doi:10.1016/s0014-5793(02)02808-9

Cited by 58 publications

(68 citation statements)

References 59 publications

(73 reference statements)

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“…While the spectra of non-fluorescent CA-GFP and fluorescent wild-type GFP are not super-imposable (Fig. 6C), the similarity in the spectral features were coincident with published spectra (33) and suggest that CA-GFP forms the ␤-barrel observed in all GFPs. The gain in the intensity of the circular dichroism signal is consistent with an increase in the multimeric state of CA-GFP.…”

Section: Ca-gfp Requires No Cofactors and Can Be Activated In Vitro-supporting

confidence: 59%

Mechanism of a Genetically Encoded Dark-to-Bright Reporter for Caspase Activity

Nicholls

Chu

Abbruzzese

et al. 2011

Journal of Biological Chemistry

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Fluorescent proteins have revolutionized modern biology with their ability to report the presence of tagged proteins in living systems. Although several fluorescent proteins have been described in which the excitation and emission properties can be modulated by external triggers, no fluorescent proteins have been described that can be activated from a silent dark state to a bright fluorescent state directly by the activity of an enzyme. We have developed a version of GFP in which fluorescence is completely quenched by appendage of a hydrophobic quenching peptide that tetramerizes GFP and prevents maturation of the chromophore. The fluorescence can be fully restored by catalytic removal of the quenching peptide, making it a robust reporter of proteolysis. We have demonstrated the utility of this uniquely dark state of GFP as a genetically encoded apoptosis reporter that monitors the function of caspases, which catalyze the fate-determining step in programmed cell death. Caspase Activatable-GFP (CA-GFP) can be activated both in vitro and in vivo, resulting in up to a 45-fold increase in fluorescent signal in bacteria and a 3-fold increase in mammalian cells. We used CA-GFP successfully to monitor real-time apoptosis in mammalian cells. This dark state of GFP may ultimately serve as a useful platform for probes of other enzymatic processes.

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Section: Ca-gfp Requires No Cofactors and Can Be Activated In Vitro-supporting

confidence: 59%

Mechanism of a Genetically Encoded Dark-to-Bright Reporter for Caspase Activity

Nicholls

Chu

Abbruzzese

et al. 2011

Journal of Biological Chemistry

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“…YFP was produced in Escherichia coli BL21(DE3) as a fusion protein with an intein-chitin binding domain (CBD) tag in plasmid vector pTYB11 as described by Visser et al (2002). The fusion protein was bound to a column of chitin beads, and the tag was removed on-column by inteinmediated splicing induced with 50 mM DTT.…”

Section: Affinity-purified Yfp Antibodiesmentioning

confidence: 99%

Endocytic and Secretory Traffic in Arabidopsis Merge in the Trans-Golgi Network/Early Endosome, an Independent and Highly Dynamic Organelle

et al. 2010

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Plants constantly adjust their repertoire of plasma membrane proteins that mediates transduction of environmental and developmental signals as well as transport of ions, nutrients, and hormones. The importance of regulated secretory and endocytic trafficking is becoming increasingly clear; however, our knowledge of the compartments and molecular machinery involved is still fragmentary. We used immunogold electron microscopy and confocal laser scanning microscopy to trace the route of cargo molecules, including the BRASSINOSTEROID INSENSITIVE1 receptor and the REQUIRES HIGH BORON1 boron exporter, throughout the plant endomembrane system. Our results provide evidence that both endocytic and secretory cargo pass through the trans-Golgi network/early endosome (TGN/EE) and demonstrate that cargo in late endosomes/multivesicular bodies is destined for vacuolar degradation. Moreover, using spinning disc microscopy, we show that TGN/EEs move independently and are only transiently associated with an individual Golgi stack.

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“…In case of ECFP the fine structure of excitation and emission bands can be clearly distinguished. This fine structure originates from vibrational transitions superimposed on the single electronic transition [1,12]. The spectra and the maximum extinction coefficients are taken as described previously [3].…”

Section: Excitation and Fluorescence Spectra On Wavenumber Scalementioning

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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Circular dichroism spectroscopy of fluorescent proteins

Cited by 58 publications

References 59 publications

Mechanism of a Genetically Encoded Dark-to-Bright Reporter for Caspase Activity

Mechanism of a Genetically Encoded Dark-to-Bright Reporter for Caspase Activity

Endocytic and Secretory Traffic in Arabidopsis Merge in the Trans-Golgi Network/Early Endosome, an Independent and Highly Dynamic Organelle

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

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