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

Abstract

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...

Subunit counting in membrane-bound proteins

“…2b). This supports the idea that ∼20% of the GFPs are in a nonfluorescent state, reminiscent of what has been observed for DsRed 15 . The confidence level that the observed distribution represents a tetramer is high.…”

A Purple-blue Chromoprotein from Goniopora tenuidens Belongs to the DsRed Subfamily of GFP-like Proteins

“…These data suggest that the conditions and the extent of fragmentation are characteristic features for a chromophore type of GFP-like proteins. The fragmentation of DsRed was shown to occur after the complete hydrolysis of an acylimine and does not exceed 50% of the total protein, because mature DsRed contains red and green species in an approximately equal ratio (14,17). As expected, DsRed, boiled in the electrophoresis buffer, showed two fragment bands in SDS/PAGE, with apparent masses of 10 kDa and 18 kDa and a major band of a full-length protein, with an M r of 28 kDa (Fig.…”

Single-molecule spectroscopy of fluorescent proteins

“…This stepwise decrease of emission intensity was attributed to the bleaching of individual chromophores within the oligomer (Fig. 4) [115][116][117], confirming the tetrameric nature of the protein.…”