DsRed proteins

About one year after its first description the three dimensional structure of DsRED and chemical structure of the its chromophore was described by two independent groups [25, 26]. The structural data and other experimental evidences proved that the DsRED protein is an obligate tetramer and that the chromophore is GFP-like but possesses an extended 7i-electron system due to an additional oxidation step [27,28]. 

Due to its unique red-fluorescence the DsRED protein gained strong interest amongst researchers, but it soon turned out that the usability of this protein as a reporter is limited by several factors its... 

Introduction of the mutations K70R, K70M,Y120H and S197T into the wildtype DsRED protein shows similar results. 

Recently a monomeric form of the DsRED protein named mRFPl was described [29]. However, this derivative of the DsRED is heavily mutated and carries 33 amino-acid exchanges. Furthermore, the spectral properties differ significantly from those... 

Mature wildtype DsRED protein and its recombinant derivatives DsRED2, DsRED.Tl, DsRED.T3 DsRED.T4 proved to be stable (soluble and fluorescent) at temperatures up to 70-75 °C, Fig. (19). For RedStar, mRFPl, Fluorescent timer , mcavRFP, HcRED and equaRFPl (eqFP611) such data currently are not available. 

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... 

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. 

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. 

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... 

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. 

---