Structural and functional relationships of plant cryptochromes

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Cryptochromes are ubiquitous UV-A/blue-light photoreceptors related to light-driven DNA-repair enzymes (DNA-photolyases), which catalyze the removal of UV-B photoproducts from DNA. Cryptochromes, though having the same cofactors FAD and methenyl-tetrahydrofolate (MTHF) as the DNA-photolyases, have no photolyase activity. The crystal structures of three bacterial photolyases (Park et al., 1995; Tamada et al., 1997; Komori et al., 2001) and one bacterial cryptochrome have been solved (Brudler et al., 2003), demonstrating that this protein family is accessible to structural analysis at the atomic level. Most recently others and we discovered that cryptochromes can directly bind to DNA. Due to the progress we have made in the heterologous expression of plant cryptochromes we want to address in this proposal the function of plant cryptochromes at the atomic level. For this we will produce and analyze two cryptochromes that are located in different compartments in the plant cell, one located in the nucleus and the other located in the organelles. The proteins will be subjected to various spectroscopic analyses including crystallization and X-ray spectroscopy. Results from spectroscopy will be used to modify cryptochromes by site-directed mutagenesis and to analyze the effects of mutation on its biological function in vivo and in vitro.