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Green fluorescent protein (GFP) and homologous proteins possess a unique pathway of chromophore formation based on autocatalytic modification of their own amino acid residues. Green-to-red photoconvertible fluorescent protein Kaede carries His–Tyr–Gly chromophore-forming triad [21].

Chromophore in GFP is formed by cyclization and further dehydration and oxidation of amino acids at positions 65–67 (Ser–Tyr–Gly). These reactions result in 4-( p -hydroxybenzylidene)-5-imidazolone, which includes a newly formed 5-membered heterocycle and Tyr66-derived phenolic ring connected by a methylene bridge ( Scheme 1 ). In all known natural GFP-like proteins positions corresponding to the chromophore-forming Tyr66 and Gly67 are invariant, while the first chromophore-forming residue 65 can vary. Studies of last several years clearly demonstrated that 4-( p -hydroxybenzylidene)-5-imidazolone chromophore core is common for all natural GFP-like proteins. At the same time, red-shifted proteins can contain a modification of the protein backbone at position 65. Two main types of red chromophores within GFP-like proteins are known. One type was first described for DsRed: chromophore consists of GFP chromophore core extended with an acylimine group ( Scheme 1 ) which is formed as a result of dehydrogenation of C a N bond of the first chromophore-forming residue with molecular oxygen [22].

Sheme 1: Origination of DsRed-type and Keade-type chromophores from GFP-type precursor in the course of posttranslational modifications of fluorescent proteins.

Yampolsky R. et al., Bioorganic Chemistry, 2008, 36, 97

This oxidation is catalyzed by nearby residues and occurs in the dark, although it can be greatly facilitated by irradiation with light at about 400 nm. Second red chromophore type is characteristic for green to red photoconvertible fluorescent proteins carrying His–Tyr–Gly chromophore-forming residues. The first protein of this type named Kaede was cloned from stony coral Trachyphyllia geoffroyi in 2002 . In the dark, Kaede is a stable green fluorescent protein with a GFP-like chromophore. However, irradiation with UV or violet light (at approximately 350–420 nm) efficiently converts Kaede into red fluorescent state. Kaede red chromophore is formed by protein backbone break between N and C a of His65. As a result, GFP-like chromophore core is brought into conjugation with His65 heterocycle ( Scheme 1 ). This reaction is non-oxidative and requires no molecular oxygen. Such mutants are either non-fluorescent or have no ability of green-to-red conversion. This failure might be a result of improper orientation of the bigger (compared to His) aromatic residues. It is known from crystal structure of Kaede-like proteins that position and orientation of the chromophore-forming His is almost unchanged during green-to-red photoconversion. Thus, possibly the chromophore environment catalyses formation of the red chromophore by stabilizing the product-like transition state. Another explanation is a direct participation of His65 imidazolering in protonation–deprotonation events during photoconversion. In this case, Phe, Tyr or Trp residues cannot act as His. Anyhow, absence of corresponding mutants of Kaede-like proteins precludes studying spectral properties of Kaede-like chromophore variants [23].

Spectral properties

Wild type Kaede chromophore HYG demonstrated clear pH dependence of absorption spectrum. In water solutions, acidic form absorbed at 430 nm. With an increase of pH, this peak transformed into a peak at 490 nm with isosbestic point at 447 nm and p K a 7.7.

FIG1: Absorption spectra for HYG chromophore. (A) Absorption spectra for buffered water solutions of HYG chromophore at different pH as designated on the graphs.

Yampolsky R. et al., Bioorganic Chemistry, 2008, 36, 102

This transition is attribuited to deprotonation of phenolic oxygen that is a common process for chromophores within other GFP-like proteins. No other spectral transitions were observed in interval pH 3–14. Nature of solvent was found to have a minor impact on absorption maximum and extinction coefficient of the acidic form of HYG ( Fig. 2 ).

FIG 2: Absorption spectra for HYGchromophore in different solvents at pH 3.5 (B) or pH 10.1

Yampolsky R. et al., Bioorganic Chemistry, 2008, 36, 103

In contrast, in basic conditions HYG chromophore possessed clearly different absorption spectra in different solvents. Strong absorption red shift was observed in the series water–ethanol–isopropanol– DMF–DMSO [24].