Picosecond Time-Resolved FRET in the Fluorescent Protein from Discosoma Red (wt-DsRed)

Schüttrigkeit, Tanja A.; Zachariae, Ulrich; Feilitzsch, Till von; Wiehler, Jens; Hummel, Julia; Steipe, Boris; Michel‐Beyerle, M. E.

doi:10.1002/1439-7641(20010518)2:5<325::aid-cphc325>3.3.co;2-g

Cited by 6 publications

(12 citation statements)

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“…Moreover, it is established from recent time-resolved fluorescence studies in vitro that oligomers comprising both immature green and mature red forms of the coral FP known as Discosoma Red (Ds-Red) exhibit FRET (41,42). The reciprocal kinetic responses observed in vitro generally correspond with those obtained here in vivo with the blue and green FP.…”

Section: Evidence For Förster Resonance Energy Transfer Among Fluoressupporting

confidence: 66%

Simultaneous Time Resolution of the Emission Spectra of Fluorescent Proteins and Zooxanthellar Chlorophyll in Reef-building Corals¶†

Gilmore

Larkum

Salih

et al. 2003

Photochem Photobiol

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Light is absorbed by photosynthetic algal symbionts (i.e. zooxanthellae) and by chromophoric fluorescent proteins (FP) in reef-building coral tissue. We used a streak-camera spectrograph equipped with a pulsed, blue laser diode (50 ps, 405 nm) to simultaneously resolve the fluorescence spectra and kinetics for both the FP and the zooxanthellae. Shallow water (<9 m)-dwelling Acropora spp. and Plesiastrea versipora specimens were collected from Okinawa, Japan, and Sydney, Australia, respectively. The main FP emitted light in the blue, blue-green and green emission regions with each species exhibiting distinct color morphs and spectra. All corals showed rapidly decaying species and reciprocal rises in greener emission components indicating Förster resonance energy transfer (FRET) between FP populations. The energy transfer modes were around 250 ps, and the main decay modes of the acceptor FP were typically 1900-2800 ps. All zooxanthellae emitted similar spectra and kinetics with peak emission (approximately 683 nm) mainly from photosystem II (PSII) chlorophyll (chl) a. Compared with the FP, the PSII emission exhibited similar rise times but much faster decay times, typically around 640-760 ps. The fluorescence kinetics and excitation versus emission mapping indicated that the FP emission played only a minor role, if any, in chl excitation. We thus suggest the FP could only indirectly act to absorb, screen and scatter light to protect PSII and underlying and surrounding animal tissue from excess visible and UV light. We conclude that our time-resolved spectral analysis and simulation revealed new FP emission components that would not be easily resolved at steady state because of their relatively rapid decays due to efficient FRET. We believe the methods show promise for future studies of coral bleaching and for potentially identifying FP species for use as genetic markers and FRET partners, like the related green FP from Aequorea spp.

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Section: Evidence For Förster Resonance Energy Transfer Among Fluoressupporting

confidence: 66%

Simultaneous Time Resolution of the Emission Spectra of Fluorescent Proteins and Zooxanthellar Chlorophyll in Reef-building Corals¶†

Gilmore

Larkum

Salih

et al. 2003

Photochem Photobiol

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“…Finally, the tendency of many fluorescent proteins to form dimers and tetramers [35,[91][92][93] can lead to complex energy-transfer interactions between the proteins within the oligomer [86,92,94].…”

Section: General Structure Of Vfpsmentioning

confidence: 99%

“…The fluorescence decay characteristics recorded on ensemble samples of DsRed are a complex mixture of a number of fast and slow decay components ranging from several hundreds of picoseconds to nanoseconds [94,134,137]. This complex decay characteristic is generally attributed to the presence of two different chromophores, one green and one red emitting, and energy-transfer coupling of these chromophores within one tetramer.…”

Section: Fret Coupling In Dsred Proteins By Multiparameter Spectroscopymentioning

confidence: 99%

Single-molecule spectroscopy of fluorescent proteins

Blum

Subramaniam

2008

Anal Bioanal Chem

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The discovery and use of fluorescent proteins has revolutionized cellular biology. Despite the widespread use of visible fluorescent proteins as reporters and sensors in cellular environments the versatile photophysics of fluorescent proteins is still subject to intense research. Understanding the details of the photophysics of these reporters is essential for accurate interpretation of the biological and biochemical processes illuminated by fluorescent proteins. Some aspects of the complex photophysics of fluorescent proteins can only be observed and understood at the singlemolecule level, which removes averaging inherent to ensemble studies. In this paper we review how singlemolecule emission detection has helped understanding of the complex photophysics of fluorescent proteins.

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“…2), which can be attributed to the chromophore protection by the b-barrel. This observation is particularly important for several applications that require accurate knowledge of the absorption and emission spectra, such as FRET (Day et al, 1999(Day et al, , 2001Jones et al, 2000;Mizuno et al, 2001;Schuttrigkeit et al, 2001;Gautier et al, 2001), dual-color cross-correlation FCS (Koltermann et al, 1998), and dual-channel imaging using 1P-and 2P-photon fluorescence microscopy, where fluorescence detection can be optimized without ambiguity. Such fluorescence detection optimization allows for minimal illumination intensities to be used to avoid photodamage of biological samples.…”

Section: Steady-state Spectroscopymentioning

confidence: 99%

“…Wild-type (wt) green fluorescent protein (GFP) and red fluorescent protein (DsRed), isolated from the jellyfish Aequorea victoria and Discosoma corallimorpharian, respectively, have become invaluable fluorescent markers for biological studies (Chalfie and Kain, 1998;Tsien, 1998;Baird et al, 2000;Gross et al, 2000;Fradkov et al, 2000;Belmont, 2001;Chiesa et al, 2001;Wahlfors et al, 2001;Ayoob et al, 2001;Day et al, 1999Day et al, , 2001Zimmer, 2002). The wide range of absorption/emission wavelengths of these intrinsically fluorescent proteins (IFPs) and their mutants allows for multicolor labeling, negligible cellular intrinsic fluorescence, and the creation of fluorescence donoracceptor pairs for Förster resonance energy transfer (FRET), a popular technique in cell biology for studying proteinprotein interactions (Day et al, 1999(Day et al, , 2001Jones et al, 2000;Mizuno et al, 2001;Schuttrigkeit et al, 2001). Genetic encoding of IFPs allows for site-specific labeling within cells for visualization of gene expression and cellular functions and opens many exciting opportunities in biological and biomedical research.…”

Section: Introductionmentioning

confidence: 99%

Quantitative Analysis of the Fluorescence Properties of Intrinsically Fluorescent Proteins in Living Cells

Hess

Sheets

Wagenknecht-Wiesner

et al. 2003

Biophysical Journal

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The main potential of intrinsically fluorescent proteins (IFPs), as noninvasive and site-specific markers, lies in biological applications such as intracellular visualization and molecular genetics. However, photophysical studies of IFPs have been carried out mainly in aqueous solution. Here, we provide a comprehensive analysis of the intracellular environmental effects on the steady-state spectroscopy and excited-state dynamics of green (EGFP) and red (DsRed) fluorescent proteins, using both one- and two-photon excitation. EGFP and DsRed are expressed either in the cytoplasm of rat basophilic leukemia (RBL-2H3) mucosal mast cells or anchored (via LynB protein) to the inner leaflet of the plasma membrane. The fluorescence lifetimes (within approximately 10%) and spectra in live cells are basically the same as in aqueous solution, which indicate the absence of both IFP aggregation and cellular environmental effects on the protein folding under our experimental conditions. However, comparative time-resolved anisotropy measurements of EGFP reveal a cytoplasmic viscosity 2.5 +/- 0.3 times larger than that of aqueous solution at room temperature, and also provide some insights into the LynB-EGFP structure and the heterogeneity of the cytoplasmic viscosity. Further, the oligomer configuration and internal depolarization of DsRed, previously observed in solution, persists upon expression in these cells. DsRed also undergoes an instantaneous three-photon induced color change under 740-nm excitation, with efficiently nonradiative green species. These results confirm the implicit assumption that in vitro fluorescence properties of IFPs are essentially valid for in vivo applications, presumably due to the beta-barrel protection of the embodied chromophore. We also discuss the relevance of LynB-EGFP anisotropy for specialized domains studies in plasma membranes.

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Picosecond Time-Resolved FRET in the Fluorescent Protein from Discosoma Red (wt-DsRed)

Cited by 6 publications

References 0 publications

Simultaneous Time Resolution of the Emission Spectra of Fluorescent Proteins and Zooxanthellar Chlorophyll in Reef-building Corals¶†

Simultaneous Time Resolution of the Emission Spectra of Fluorescent Proteins and Zooxanthellar Chlorophyll in Reef-building Corals¶†

Single-molecule spectroscopy of fluorescent proteins

Quantitative Analysis of the Fluorescence Properties of Intrinsically Fluorescent Proteins in Living Cells

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