Green fluorescent protein from Aequorea victoria and it's homologs have been cloned from various marine organism. A number of optimized blue, cyan, and yellow mutants of GFP have also been developed. Therefore, GFP has been used extensively as a genetically encodable fluorescent probe in molecular and cell biology. A wide variety of different GFP variants hace been constructed using genetic engineering techniques, and a number of mutation in and around the chromopore have been shown to have profound effects on the protein's optical properties.

The blue fluorescent protein (BFP) variants are characterized by moderate intrinsic brightness (defined as the product of extinction coefficient and quantum yield) and low photostability. Recently, two improved BFP variants, Azurite and EBFP2, were introduced. Both Azurite and EBFP2 exhibited significant improvement in photostability compared with original BFP and EBFP2 [25].

The researchers used a red chromophore formation pathway, in which that anionic red chromophore is formed from the neutral blue intermediate,to suggest a rational design strategy to develop blue fluorescent proteins with a tyrosine-based chromophore. Among five different BFP variants obtained by this strategy, enhaced mTagBFP protein has the intrinsic brightness similar to that of EGFP; the higher pH-stability makes mTagBFP suitable for visualization in acid compartments.

The strategy was applied to red fluorescent proteins of the different genetic backgrounds, such as TagRFP, mCherry, HcRed1, M355NA, and mKeima,which all were converted into blue probes. Further improvement of the blue variant of TagRFP by random mutagenesis resulted in an enhanced monomeric protein,mTagBFP, characterized by the substantially higher brightness, the faster chromophore maturation, and the higher pH stability than blue fluorescent proteins with a histidine in the chromophore. The detailed biochemical and photochemical analysis indicates that mTagBFP is the true monomeric protein tag for multicolor and lifetime imaging, as well as the outstanding donor for green fluorescent protein in resonance energy transfer applications.

The improvement in the brightness was mainly due to the increase in the quantum yields, but not in the molar extinction coefficient. Because all BFPs have His66 in the chromophore, the researchers pseculated that their low absorbance would be attibuite to this residue, and it's change to Tyr66, which is tipically observed in green and yellow variants, such as EGEP and EYFP, could increase the extinction coefficient. Therefore, they turned our attention to red fluorescent proteins (RFPs) with anionic chromophore, where ESPT has not observed. Previously, they have shown that the DsRed-like red chromophore is mainly formed from a blue protonated GFP-like chromophore as an intermediate.

Here, the researchers use a structure-based directed evolution in combination with random mutagenesis to develop monomeric BFPs on the basis of the RFPs of different genetic background, such as TagRFP, mCherry, HcRed1, M355NA, and Mkeima [26].

They selected several monomeric RFPs, TagRFp,ecc..., to introduce site specific mutation preventing maturation of the chromophore beyond the blue protonated intermediate, and also stabilizing it. Their primary choice of positions for amino acid substitutions was based on several crystal structures:

FIG 1: cromophpre of blu fluorescence protein

Subach O. M., Gundorov I. S., Yoshimura M., Subach F. V.,1 Zhang Y., Chemistry & Biology, 2008, 15, 1117