DsRed mutants

The fluorescent timer is another DsRED mutant with the unique ability to change its light emission peaks from green to red over time. This property results from the pointmutations VI05A and S197T present in fluorescent timer [32],... 

In summary, DsRed and its mutants represent interesting fusion partners for in vivo cross-correlation analysis although drawbacks cannot be excluded. Nevertheless, researches should consider the variety of DsRed mutants existing to date and evaluate compromises such as monomeric but rather dark (mRFPl) or bright but bulky dimeric tags such as tdimer2(12). 

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. 

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]. 

However, during the last decade a number of GFP mutants were described showing altered spectral properties and/or improved solubility upon expression in heterologous systems. In addition, numerous other naturally occurring fluorescent proteins were described such as the red-fluorescent protein from Discosoma sp. (DsRED). A comprehensive description of the available fluorescent proteins is given including the spectral properties, amino-acid sequence alignments, comparisons of the secondary and tertiary structures of the proteins. 

As a further drawback, native DsRed also exhibits slow and complex fluo-rophore maturation due to an additional autocatalytic modification, extending the chromophore s conjugation system and allowing for red fluorescence [44, 45]. Non-mature protein with 475-nm excitation/500-nm emission maxima transforms into mature protein with 558-nm excitation/585-nm emission maxima and requires >48 h to reach 90% of maximal fluorescence [39]. Fluo-rophore maturation has been significantly accelerated in a nimiber of mutants, e.g. Tl, mRFPl and E57 and was proposed to depend upon the space arovmd the fluorophore [46,47]. 

A new red protein eqFP611 from Entacmaea quadricolor displayed both monomeric and tetrameric features in initial studies [78]. FCS analysis at low nmol/1 concentrations predicted a mass ratio of 2.7 between DsRed and eqFP611 and produced a diffusion coefficient similar to the monomeric GFP mutant Citrine. In contrast, size-exclusion chromatography and the recently resolved crystal structure of eqFP611 indicated that eqFP611 too can form tetramers... 

DsRed and its mutants display emission spectra sufficiently red-shifted for two-colour applications with GFP mutants. The only combination of GFP mutants to display a similar small spectral overlap is presented by EBFP and EYFP, but no FCS application of EBFP has been reported so far and its excitation max-imiun (388 nm) is unfavourable for intracellular applications. 

Proteins that exhibit fluorescence in their native form were extensively studied and utilized in a variety of apphcations. One important example is green fluorescent protein (GFP) of Aequorea victoria and its mutants (BFP, CFP, YFP), which exhibit blue, cyan, and yeUow emissions, respectively. In addition, a novel fluorescent protein, termed DsRed, was recently cloned and characterized. These proteins are unique in having fluorophores formed from the natural amino acid side chains via cyclization. A major application for them is the creation and expression of fluorescent fusion proteins. Such fusion constructs were used in a variety of apphcations involving in vitro and in vivo spatial and temporal fluorescence... 

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