Structural and functional relationships of plant cryptochromes

隐花色素是普遍存在的UV-A/蓝光光感受器，与光驱动的DNA修复酶（DNA光解酶）相关，其催化从DNA去除UV-B光产物。隐花色素虽然具有与DNA光解酶相同的辅因子FAD和亚甲基四氢叶酸（MTHF），但没有光解酶活性。三种细菌光解酶的晶体结构（Park等人，1995; Tamada等人，1997; Komori等人，2001）和一种细菌隐花色素（Brudler等人，2003年），表明该蛋白质家族可在原子水平上进行结构分析。最近，我们和其他人发现隐花色素可以直接与DNA结合。由于我们在植物隐花色素的异源表达方面取得的进展，我们希望在本提案中解决植物隐花色素在原子水平上的功能。为此，我们将产生和分析两种隐花色素，它们位于植物细胞的不同隔室中，一种位于细胞核中，另一种位于细胞器中。蛋白质将进行各种光谱分析，包括结晶和X射线光谱。从光谱学的结果将被用来修改隐花色素的定点突变，并分析其在体内和体外的生物功能的突变的影响。

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.