Far-red fluorescent proteins evolved from a blue chromoprotein from Actinia equina.

Proteins of the GFP (green fluorescent protein) family demonstrate a great spectral and phylogenetic diversity. However, there is still an intense demand for red-shifted GFP-like proteins in both basic and applied science. To obtain GFP-like chromoproteins with red-shifted absorption, we performed a broad search in blue-coloured Anthozoa species. We revealed specimens of Actinia equina (beadlet anemone) exhibiting a bright blue circle band at the edge of the basal disc. A novel blue chromoprotein, aeCP597, with an absorption maximum at 597 nm determining the coloration of the anemone basal disk was cloned. AeCP597 carries a chromophore chemically identical with that of the well-studied DsRed (red fluorescent protein from Discosoma sp.). Thus a strong 42-nm bathochromic shift of aeCP597 absorption compared with DsRed is determined by peculiarities of chromophore environment. Site-directed and random mutagenesis of aeCP597 resulted in far-red fluorescent mutants with emission maxima at up to 663 nm. The most bright and stable mutant AQ143 possessed excitation and emission maxima at 595 and 655 nm respectively. Thus aeCP597 and its fluorescent mutants set a new record of red-shifted absorption and emission maxima among GFP-like proteins.

Chromophore environment provides clue to "kindling fluorescent protein" riddle.

asCP, the unique green fluorescent protein-like nonfluorescent chromoprotein from the sea anemone Anemonia sulcata, becomes fluorescent ("kindles") upon green light irradiation, with maximum emission at 595 nm. The kindled protein then relaxes to a nonfluorescent state or can be "quenched" instantly by blue light irradiation. In this work, we used asCP mutants to investigate the mechanism underlying kindling. Using site-directed mutagenesis we showed that amino acids spatially surrounding Tyr(66) in the chromophore are crucial for kindling. We propose a model of the kindling mechanism, in which the key event is chromophore turning or cis-trans isomerization. Using site-directed mutagenesis we also managed to transfer the kindling property to the two other coral chromoproteins. Remarkably, most kindling mutants were capable of both reversible and irreversible kindling. Also, we obtained novel variants that kindled upon blue light irradiation. The diversity of photoactivated fluorescent proteins that can be developed by site-directed mutagenesis is promising for biotechnological needs.

Interconversion of Anthozoa GFP-like fluorescent and non-fluorescent proteins by mutagenesis.

BACKGROUND: Within the family of green fluorescent protein (GFP) homologs, one can mark two main groups, specifically, fluorescent proteins (FPs) and non-fluorescent or chromoproteins (CPs). Structural background of differences between FPs and CPs are poorly understood to date. RESULTS: Here, we applied site-directed and random mutagenesis in order to to transform CP into FP and vice versa. A purple chromoprotein asCP (asFP595) from Anemonia sulcata and a red fluorescent protein DsRed from Discosoma sp. were selected as representatives of CPs and FPs, respectively. For asCP, some substitutions at positions 148 and 165 (numbering in accordance to GFP) were found to dramatically increase quantum yield of red fluorescence. For DsRed, substitutions at positions 148, 165, 167, and 203 significantly decreased fluorescence intensity, so that the spectral characteristics of these mutants became more close to those of CPs. Finally, a practically non-fluorescent mutant DsRed-NF was generated. This mutant carried four amino acid substitutions, specifically, S148C, I165N, K167M, and S203A. DsRed-NF possessed a high extinction coefficient and an extremely low quantum yield (< 0.001). These spectral characteristics allow one to regard DsRed-NF as a true chromoprotein. CONCLUSIONS: We located a novel point in asCP sequence (position 165) mutations at which can result in red fluorescence appearance. Probably, this finding could be applied onto other CPs to generate red and far-red fluorescent mutants. A possibility to transform an FP into CP was demonstrated. Key role of residues adjacent to chromophore's phenolic ring in fluorescent/non-fluorescent states determination was revealed.