Isolation of Drosophila and Mouse Cryptochromes for Structural, Biochemical and Spectroscopic Investigations
Final Report Abstract
Drosophila cryptochrome (dCRY) is an FAD binding blue-light photoreceptor mediating light synchronization of the circadian clock. The mouse cryptochromes (mCRY1/2) are involved in the transcriptional regulation of the mammalian circadian clock. The aim of this project was to study the structure, photochemistry and molecular interactions of dCRY and mCRY1/2 using a combination of X-ray crystallography, biochemical and spectroscopic techniques. We have succeeded in expressing and purifying mg amounts of dCRY, mCRY1/2 and C-terminal mCRY-CCtail fragments. Purified dCRY was used for crystallization and analysis of its photoreaction mechanism by UV/VIS- and EPR spectroscopy. Further insights into the dCRY photoreaction were obtained by analysing dCRY mutants in S2 Drosophila Schneider cells (proteasomal degradation) and by UV/VIS spectroscopy (formation and decay of FAD°-). Purified mCRY proteins were used for interaction studies (fluorescence polarisation, isothermal titration calorimetry, analytical ultracentrifugation, peptide scan) with the transcription factor mBMAL1.
Publications
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A novel Photoreaction Mechanism for the circadian Blue-Light Photoreceptor Drosophila Cryptochrome. JBC 2007 April 27; 282(17): 13011-13021
Berndt A., Kottke T., Breitkreuz H., Dvorsky R., Hennig S., Alexander M. and Wolf E.
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Protein Aggregation Studied by Forward Light Scattering and Light Transmission Analysis. Chemical Physics 2007 Dec 6; 342(1): 55-63
Penzkofer A., Shirdel J., Zirak P., Breitkreuz H. and Wolf E.
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Fluorescence spectroscopic Characterization of the circadian blue-light Photoreceptor Cryptochrome from Drosophila melanogaster. Chemical Physics 2008 Aug 28; 352(1): 35-47
Shirdel J., Zirak P., Penzkofer A., Breitkreuz H. and Wolf E.
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Human and Drosophila Cryptochromes are light activated by Flavin Photoreduction in living Cells. PLoS Biology 2008 Jul 1;6(7):e160
Hoang N., Schleicher E., Kacprzak S., Bouly JP, Picot M., Wu W., Berndt A., Wolf E., Bittl R. and Ahmad M.
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Degradation of (6R,S)-5,10-Methenyltetrahydrofolate in aqueous Solution at pH 8 and pH 2.5. Chemical Physics 2009, 358 (1-2), 132-136
Tyagi A., Penzkofer A., Batschauer A. and Wolf E.
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Fluorescence Behaviour of 5,10-Methenyltetrahydrofolate, 10-Formyltetrahydrofolate, 10-Formyldihydrofolate, and 10- Formylfolate in Aqueous Solution at pH 8. Chemical Physics 2009, 361 (1-2), 75-82
Tyagi A., Penzkofer A., Batschauer A. and Wolf E.
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Quantitative analyses of Cryptochrome - mBMAL1 interactions: mechanistic insights into the transcriptional regulation of the mammalian circadian clock. JBC, 2011 Jun 24;286(25):22414-25
Czarna, A., Breitkreuz H., Mahrenholz C.C., Arens J., Strauss H.M. and Wolf E.
