Isolation of Drosophila and Mouse Cryptochromes for Structural, Biochemical and Spectroscopic Investigations

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Cryptochromes are FAD- and MTHF binding proteins. Drosophila Cryptochrome (dCry) is a blue-light photoreceptor involved in synchronization of the circadian (24h) rhythm with the environmental dark-light cycle. The two mouse Cryptochromes (mCry 1 and 2) act as integral components of the mouse circadian oscillator in a light-independent manner. The goal of this research proposal is to study the structures and photochemistry of Drosophila and mouse Cryptochromes and their complexes with effector molecules using a combination of X-ray crystallography, biochemical and spectroscopic techniques. We are using E.coli and SF21 insect cells for expression and coexpression of Cryptochromes and interacting clock components. Purified Drosophila and mouse Cryptochromes alone or in a complex with interacting dTim and mPer fragments will be used for crystallisation and structure determination by x-ray crystallography. Photochemical studies (ESR, vibrational and ultrafast spectroscopy) and time-resolved crystallography will be performed in collaborating groups of the consortium (R. Bittl, J. Heberle, A. Penzkofer, I.Schlichting). In collaboration with A. Batschauer, we will pursue the crystallisation and crystal structure determination of the A.thaliana Cryptochromes At-Cry2 and At-Cry3.