Identification of different emitting species in the red fluorescent protein DsRed by means of ensemble and single-molecule spectroscopy

105

101

Recent studies on the newly cloned red fluorescence protein DsRed from the Discosoma genus have shown its tremendous advantages: bright red fluorescence and high resistance against photobleaching. However, it has also become clear that the protein forms closely packed tetramers, and there is indication for incomplete protein maturation with unknown proportion of immature green species. We have applied single-molecule methodology to elucidate the nature of the fluorescence emission in the DsRed. Real-time fluorescence trajectories have been acquired with polarization sensitive detection. Our results indicate that energy transfer between identical monomers occurs efficiently with red emission arising equally likely from any of the chromophoric units. Photodissociation of one of the chromophores weakly quenches the emission of adjacent ones. Dual color excitation (at 488 and 568 nm) single-molecule microscopy has been performed to reveal the number and distribution of red vs. green species within each tetramer. We find that 86% of the DsRed contain at least one green species with a red-to-green ratio of 1.2-1.5. On the basis of our findings, oligomer suppression would not only be advantageous for protein fusion but will also increase the fluorescence emission of individual monomers.T he cloning of the red fluorescent protein (drFP583, commercially available as DsRed) from the Indo pacific reef coral Discosoma sp (1) has triggered intense biological interest as a potential expression marker and fusion partner that would be complementary to the Aequorea victoria green fluorescent protein (avGFP). The main advantage of these naturally fluorescent proteins is that they provide strong visible fluorescence and can be genetically fused to other proteins. Since the cloning of the avGFP (2, 3), different mutants have been produced to extend the palette of available colors. However, no GFP mutant has been produced with emission maxima longer than 529 nm (3). The newly cloned DsRed has bright red fluorescence with emission maxima at 583 nm, one of the longest wavelength emissions reported so far in a wild-type species [Fradkov et al. (4) have reported a wild type highly homologous to the drFP583 with 593-nm emission and a hybrid with 616-nm emission]. Potentially, DsRed is an ideal partner of GFP in fluorescence resonance energy transfer (FRET) experiments, particularly in cell biological applications in which cellular autofluorescence poses a problem (5).In the last few months, extensive research has been done in the biochemical and photophysical investigation of DsRed. The absorption spectrum of the mature protein exhibits a strong band with a peak at 558 nm and two minor shoulders at 526 and 490 nm (6). An extinction coefficient of 75,000 mol Ϫ1 ͞cm Ϫ1 and a fluorescence quantum yield of 0.7 at 558-nm excitation wavelength have been reported (7), much higher than initially published by Matz et al. (1). The protein proves to be stable under harsh pH conditions and is extremely resistant to photobleaching (7, 8). However, t...

Section: Ensemble Photobleaching Experimentssupporting

confidence: 94%

The nature of fluorescence emission in the red fluorescent protein DsRed, revealed by single-molecule detection

García-Parajo

Koopman

Dijk

et al. 2001

105

101

“…The fluorescence quantum yield of eqFP611 of 0.45 is somewhat lower than the value of 0.7 reported for DsRed (9,26). The fluorescence lifetime of eqFP611 is 2.5 ns and hence smaller than the value of 3.6 ns from ensemble measurements on DsRed (22,26), but similar to fluorescence decays measured on ''super red'' individual DsRed tetramers (27). The photobleaching quantum yield of eqFP611 determined here by single-molecule experiments, ⌽ B ϭ 3.5 ϫ 10 Ϫ6 , fits well into the range reported for DsRed by different methods, 0.8-9.5 ϫ 10 Ϫ6 (9,22,26).…”

Section: Discussioncontrasting

confidence: 48%

A far-red fluorescent protein with fast maturation and reduced oligomerization tendency from Entacmaea quadricolor (Anthozoa, Actinaria)

Wiedenmann

Schenk

Röcker

et al. 2002

236

264

We performed the biochemical and biophysical characterization of a red fluorescent protein, eqFP611, from the sea anemone Entacmaea quadricolor cloned in Escherichia coli. With an excitation maximum at 559 nm and an emission maximum at 611 nm, the recombinant protein shows the most red-shifted emission and the largest Stokes shift of all nonmodified proteins in the green fluorescent protein family. The protein fluoresces with a high quantum yield of 0.45, although it resembles the nonfluorescent members of this protein class, as inferred from the absence of the key amino acid serine at position 143. Fluorescence is constant within the range pH 4 -10. Red fluorophore maturation reaches a level of 90% after Ϸ12 h by passing through a green intermediate. After complete maturation, only a small fraction of the green species (less than 1%) persists. The protein has a reduced tendency to oligomerize, as shown by its monomeric appearance in SDS͞ PAGE analysis and single-molecule experiments. However, it forms tetramers at higher concentrations in the absence of detergent. Fluorescence correlation spectroscopy reveals light-driven transitions between bright and dark states on submillisecond and millisecond time scales. Applicability of eqFP611 for in vivo labeling in eukaryotic systems was shown by expression in a mammalian cell culture.G reen fluorescent protein from Aequorea victoria (avGFP) and its derivatives have become extremely popular in life science research as protein labels, markers of gene expression, and reporters of environmental conditions in living cells (1-3). Molecular properties of avGFP have been modified by sitedirected mutagenesis to optimize their applicability. Still, some properties, most importantly the restricted range of emission wavelengths, pose severe limitations in certain applications. In particular, red fluorescent proteins (FP) would be highly desirable because of the reduced cellular autofluorescence in the red spectral range and for use in multicolor labeling or fluorescence resonance energy transfer experiments. Efforts to produce stable red-fluorescent avGFP variants have not yet met with success. Recently, however, avGFP homologs were discovered in nonbioluminescent Anthozoa species (4-8). Some of the Anthozoa FPs show spectral properties that deviate greatly from avGFP, including red-emitting variants.One of them, a red FP from the corallimorpharian Discosoma sp. named drFP583 (6), commercially known as DsRed, became the subject of various biochemical and biophysical studies (9-12). These studies revealed detrimental properties for certain applications of the wild-type protein, especially the slow and incomplete maturation of the fluorophore, the obligate formation of tetramers, and the tendency to aggregate. Meanwhile, maturation properties have been greatly improved by engineering the protein, and the tendency to form aggregates was also reduced by mutagenesis (13-15). Recently, the oligomerization problem of DsRed has been solved by mutagenetic means (16).We have continued ...

“…ex, Excitation; em, emission; QY, quantum yield. (26). By contrast, eqFP611 was monomeric under the conditions of single-molecule experiments on immobilized preparations (9,17).…”

Section: -[(1e)-2-(5-imidazolyl)ethenyl]-4-(p-hydroxybenzylidene)-5-imentioning

confidence: 85%

EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion

Wiedenmann

Ivanchenko

Oswald

et al. 2004

634

674

A gene encoding a fluorescent protein from the stony coral Lobophyllia hemprichii has been cloned in Escherichia coli and characterized by biochemical and biophysical methods. The protein, which we named EosFP, emits strong green fluorescence (516 nm) that changes to red (581 nm) upon near-UV irradiation at Ϸ390 nm because of a photo-induced modification involving a break in the peptide backbone next to the chromophore. Single-molecule fluorescence spectroscopy shows that the wild type of EosFP is tetrameric, with strong Fö rster resonance coupling among the individual fluorophores. We succeeded in breaking up the tetramer into AB and AC subunit dimers by introducing the single point mutations V123T and T158H, respectively, and the combination of both mutations yielded functional monomers. Fusion constructs with a variety of proteins were prepared and expressed in human cells, showing that normal biological functions were retained. The possibility to locally change the emission wavelength by focused UV light makes EosFP a superb marker for experiments aimed at tracking the movements of biomolecules within the living cell.