DsRED maturation

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. 

Unlike Aequorea GFP and its derivatives the folding/maturation of DsRED does not suffer from thermosensitivity (Jach, unpublished results). However, while working with this protein it quickly turned out that the maturation-rate of this protein is extremely slow. At room-temperature the wildtype DsRED needs 24-36 hours to gain maximum fluorescence [28]. For HcRED maturation rates comparable to GFP have been published. For the other naturally occurring RFPs (see table 8) no such data are available yet. 

Under appropriate conditions it is possible to produce and isolate almost completely immature DsRED from bacterial expression systems. In contrast to flow cytometry and other whole cell approaches this allows for direct measurement of the maturation rate of the protein without ongoing protein synthesis. Interestingly, it was found that the maturation of DsRED is temperature dependent with the maturation rates increasing with temperatures, Fig. (17). At 37 °C maturation is already about threefold faster and at 60 °C the wildtype matures within 1 hour. This indicates that maturation of the wildtype protein is ineffective, but not principally limited to low rates. 

Experiments investigating cell free maturation of immature DsRED under pH conditions ranging from mild acid (pH 5.1) to... 

Unlike the other GFP-like proteins mature DsRED and its isoform shows only little pH-dependence displaying maximum fluorescence over a very broad range of pH values ranging from about 5 to 10, Fig. (19). The same holds true for equaRFPl (eqFP611) [35]. The pH-dependence of the other RFPs remains to be determined. 

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. 

T1 554/586 30,100 42 - 36% brightness relative to DsRedl - DsRed- Express - Maturation half-time 0.7 h - Tetramer like DsRedl Orange- red [46]... 

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]. 

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