DsRED fluorescence

Recently, a photoactivatable variant from Aequoria victoria green fluorescent protein (pa-GFP) was reported (Patterson and Lippincott-Schwartz 2002), yielding an increase in fluorescence emission intensity (at k 520 nm) by a factor of 100 when excited at k 488 nm after spectral activation at A. 408 nm. This phenomenon is due to an internal photoconversion process in the protein and allows spectral photoactivation of this protein in a very local way such as in the nucleus of a living cell (Post et al. 2005). In tobacco BY-2 protoplasts, we transiently co-expressed pa-GFP or pa-GFP fusion proteins and red-fluorescent protein (DsRed)-tagged prenylated Rab acceptor 1 (Pral At2g38360), a membrane protein that localizes in speckles around the nuclear envelope. The DsRed transfection allows proper cell identification and visualization before activation (via Pral -DsRed fluorescence). After pa-GFP... 

Other naturally occurring fluorescent proteins have been found in aquatic organisms including reef corals and sea anemones. The red fluorescent protein DsRed was discovered in the Anthozoan genus Discosoma. The DsRed fluorescent protein fluorophore features an imidazoline ring system similar to the GFP fluorophore but... 

Microscopy Fluorescence stereoscope, for example ZEISS Discovery V8 with mCherry or dsRED fluorescence filter set (Carl Zeiss AG, Oberkochen, Germany). 

Baird, G. S., Zacharias, D. A., and Tsien, R. Y. (2000). Biochemistry, mutagenesis, and oligomerism of DsRed, a red fluorescent protein from coral. Proc. Natl. Acad. Sci. USA 97 11984-11989. 

Gross LA, Baird GS, Hoffman RC, Baldridge KK, Tsien RY (2000) The structure of the chromophore within DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci USA 97 11990-11995... 

Wall MA, Socolich M, Ranganathan R (2000) The structural basis for red fluorescence in the tetrameric GFP homolog DsRed. Nat Stmct Biol 7 1133-1138... 

Habuchi S, Cotlet M, Gensch T, Bednarz T, Haber-Pohlmeier S, Rozenski J, Dirix G, Michiels J, Vanderleyden J, Heberle J, De Schryver FC, Hofkens J (2005) Evidence for the isomerization and decarboxylation in the photoconversion of the red fluorescent protein DsRed. J Am Chem Soc 127 8977-8984... 

The second major breakthrough for the application of fluorescent proteins was the isolation of the red fluorescent protein (RFP) drFP583 or DsRed from the Anthozoa and Discosoma sp., a mushroom-shaped anemone found in the warm waters of the Indo-Pacific ocean [13], The breakthrough was not only the discovery of the first true RFP, but equally important was the fact that it was discovered in a nonbioluminescent species and that the gene was cloned immediately. 

DsRed (Fig. 5.3E) is a bright RFP with excitation and emission maxima at 558 and 583 nm, respectively. Despite the bright red fluorescence, application of DsRed has been restricted, because of slow and inefficient maturation and its tetrameric structure [70, 71], The poor maturation efficiency has been overcome by random mutagenesis, which resulted in the fast maturing variant DsRedTl [72]. However, DsRedTl remains tetrameric. 

PAmRFPl is a variant of the RFP DsRed and mRFPl [94], Upon irradiation with 380 nm light, PAmRFPl displays a 70-fold increase in red fluorescence. However, use of PAmRFPl is limited, due to its dim red fluorescence. 

Lauf, U., Lopez, P. and Falk, M. M. (2001). Expression of fluorescently tagged connexins A novel approach to rescue function of oligomeric DsRed-tagged proteins. FEBS Lett. 498, 11-5. 

Similarly to dyes, some fluorescent proteins can be incorporated into polymeric beads to be used as an alternative for ion sensing. For example, a reporter protein (composed of a phosphate-binding protein, a FRET donor (cyan fluorescent protein) and a FRET acceptor (yellow fluorescent protein)) was incorporated into polyacrylamide nanobeads by Sun et al. [46]. FRET was inhibited upon binding of phosphate. Kopelman and co-workers [47] used a similar approach to design a nanosensor for copper ions. They have found that fluorescence of red fluorescent protein DsRed (commonly used as a label) is reversibly quenched by Cu2+ and Cu+. Both DsRed and Alexa Fluor 488 (used as a reference) were entrapped into polyacrylamide nanobeads. Typically, up to 2 ppb of copper ions can be reliably measured. It should be mentioned, that in contrast to much more robust dyes, mild conditions upon polymerization and purification are very important for immobilization of the biomolecule to avoid degradation. 

On the other hand, translocation of the MYB transcription factor LCLl fused to pa-GFP from the nucleus to the cytoplasm and its intracellular localization dynamics depends on facilitated nuclear export versus facilitated nuclear import. Protoplasts co-transfected with At2g38360-DsRed and pa-GFP-LCLl were subjected to the 2P-activation procedure and the decrease of nuclear fluorescence intensity was monitored... 

Sumner JP, Westerberg NM, Stoddard AK, Fierke CA, Kopelman R. Cu+- and Cu +-sensitive PEBBLE fluorescent nanosensors using DsRed as the recognition element. Sensors Actuators B-Chem. 2006 113 760-767. 

Single-molecule photophysics of DsRed, a red fluorescent protein [119]... 

A.A. Heikal, S.T. Hess, G.S. Baird, R.Y. Tsien, W.W. Webb, Molecular spectroscopy and dynamics of intrinsically fluorescent proteins Coral red (dsRed) and yellow (Citrine). PNAS 97(22), 11996-12001 (2000)... 

Hsiao YW, Sanchez-Garcia E, Doerr M, Thiel W (2010) Quantum refinement of protein structures implementation and application to the red fluorescent protein DsRed.Ml. J Phys ChemB 114 15413-15423... 

Examples of fluorescence labels for hgands are carboxyfluorescein, Cy3, a commercially available fluorescent marker based on a cyanine dye or tetramethyl-rhodamine. They are chemically introduced into a ligand. As with the radioactive labels, a possible influence of the labels on the binding behavior of the labeled hgands has to be considered, especially as the fluorescent dyes are complex molecules. Furthermore, the receptors themselves can be fluorescent labeled, which is done recombinantly. The respective receptors are expressed as fusion proteins with fluorescent proteins, e.g., green fluorescent protein (GFP) from Aequorea victoria, one of its mutant variants, or DSRed from Discosoma striata [26, 35]. 

Lukyanov et al. [56] have also proposed that CTI can occur in some GFP-like proteins, where it leads to a dark nonfluorescent state. They based their CTI model on some GFP-like proteins they have isolated. The majority of GFP-like proteins, such as DsRed, are fluorescent and have been isolated from corals. However, there are some nonfluorescent proteins that are in the so-called chromo state ( The chromo state indicates that the protein has a high extinction coefficient but a low quantum yield, whereas in the fluorescent state the protein is characterized by a high quantum yield. ) [56], Most interesting of these is asCP, a unique nonfluorescent GFP-like protein discovered in the sea anemone Anemonia sulcata [57]. Initially nonfluorescent, asCP can be made to fluoresce (kindled) by intense green light irradiation. After kindling the protein relaxes back to its nonfluorescent state, or it can be quenched instantly by short blue light irradiation. 

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