DsRed

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. 

Remarkably, although there is little sequence homology between the members of the GFP super family (DsRed and avGFP share less then 30% sequence homology), their crystal structures are highly similar [25-28]. The /l-barrel structure is a feature common to all members of the GFP super family for which the crystal structure has been solved. However, whereas avGFP is present mainly as a monomer, many other VFPs form obligate di- or tetramers. 

Fig. 5.2. Chromophore formation in avGFP and DsRed. Chromophore formation in avGFP (A) requires folding of the tripeptide into the right conformation in order to enable cyclization and oxidation to form the mature green chromophore. In DsRed (B) chromophore formation follows the same path as for avGFP but requires an additional oxidation step to extend the conjugation of the chromophore.

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. 

Bayle, V., Nussaume, L. and Bhat, R. A. (2008). Combination of novel GFP mutant TSapphire and DsRed variant mOrange to set up a versatile in planta FRET-FLIM assay. Plant Physiol 148, 51-60. 

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. 

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... 

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... 

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