Abstract
Cryptochromes are flavoproteins that are evolutionary related to the DNA photolyases but lack DNA repair activity. Drosophila cryptochrome (dCRY) is a blue light photoreceptor that is involved in the synchronization of the circadian clock with the environmental light-dark cycle. Until now, spectroscopic and structural studies on this and other animal cryptochromes have largely been hampered by difficulties in their recombinant expression. We have therefore established an expression and purification scheme that enables us to purify mg amounts of monomeric dCRY from Sf21 insect cell cultures. Using UV-visible spectroscopy, mass spectrometry, and reversed phase high pressure liquid chromatography, we show that insect cell-purified dCRY contains flavin adenine dinucleotide in its oxidized state (FAD(ox)) and residual amounts of methenyltetrahydrofolate. Upon blue light irradiation, dCRY undergoes a reversible absorption change, which is assigned to the conversion of FAD(ox) to the red anionic FAD(.) radical. Our findings lead us to propose a novel photoreaction mechanism for dCRY, in which FAD(ox) corresponds to the ground state, whereas the FAD(.) radical represents the light-activated state that mediates resetting of the Drosophila circadian clock.
Highlights
Drosophila cryptochrome is a blue light photoreceptor that is involved in the synchronization of the circadian clock with the environmental light-dark cycle
Using UV-visible spectroscopy, mass spectrometry, and reversed phase high pressure liquid chromatography, we show that insect cell-purified Drosophila cryptochrome (dCRY) contains flavin adenine dinucleotide in its oxidized state (FADox) and residual amounts of methenyltetrahydrofolate
Maltose-binding protein fused dCRY expressed in E. coli was shown to contain oxidized FAD, whereas MTHF binding could not be established [37, 38]
Summary
Allow for dCRY-dTIM interactions and subsequent dTIM and dCRY degradation. To date, no spectroscopic data on the blue light responses of dCRY or any other animal cryptochrome are available. To study the photochemistry of dCRY as a circadian blue light photoreceptor, we have established an expression and purification scheme that enables us to purify mg amounts of monomeric dCRY from Sf21 insect cell cultures. We show that insect cell purified dCRY contains FAD in its oxidized state (FADox) and residual amounts of MTHF. DCRY undergoes a reversible absorption change upon blue light irradiation, which is assigned to the conversion of FADox to an anionic FAD. These findings lead us to propose a novel photoreaction mechanism for dCRY, which is expected to have crucial implications for the resetting mechanism of the Drosophila circadian clock
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