The property of photoconversion in Kaede is contributed by the tripeptide, His-Tyr-Gly, that acts as a green chromophore that can be converted to red. Once Kaede is synthesized, a chromophore, 4-(p-hydroxybenzylidene)-5-imidazolinone, derived from the tripeptide mediates green fluorescence in Kaede. When exposed to UV, Kaede protein undergoes unconventional cleavage between the amide nitrogen and the α carbon (Cα) at His62 via a formal β-elimination reaction. Followed by the formation of a double bond between His62-Cα and –Cβ, the π-conjugation is extended to the imidazole ring of His62. A new chromophore, 2-[(1E)-2-(5-imidazolyl)ethenyl]-4-(p-hydroxybenzylidene)-5-imidazolinone, is formed with the red-emitting property.
The cleavage of the tripeptide was analysed by SDS-PAGE analysis. Unconverted green Kaede shows one band at 28 kDa, whereas two bands at 18 kDa and 10 kDa are observed for converted red Kaede, indicating that the cleavage is crucial for the photoconversion.
A shifting of the absorption and emission spectrum in Kaede is caused by the cleavage of the tripeptide. Before the photoconversion, Kaede displays a major absorption wavelength maximum at 508 nm, accompanied with a slight shoulder at 475 nm. When it is excited at 480 nm, green fluorescence is emitted with a peak of 518 nm.
When Kaede is irradiated with UV or violet light, the major absorption peak shifts to 572 nm. When excited at 540 nm, Kaede showed an emission maximum at 582 nm with a shoulder at 627 nm and the 518-nm peak. Red fluorescence is emitted after this photoconversion.
The photoconversion in Kaede is irreversible. Exposure in dark or illumination at 570 nm cannot restore its original green fluorescence. A reduced fluorescence is observed in red, photoconverted Kaede when it is intensively exposed to 405 nm light, followed by partial recover after several minutes.