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

隐花色素是FAD和MTHF结合蛋白。果蝇隐花色素（dCry）是一种蓝光光感受器，参与昼夜（24小时）节律与环境暗光周期的同步。两种小鼠隐花色素（mCry 1和2）以不依赖于光的方式作为小鼠昼夜节律振荡器的组成部分。该研究计划的目标是研究果蝇和小鼠隐花色素及其与效应分子的复合物的结构和光化学，使用X射线晶体学，生物化学和光谱技术的组合。我们正在使用大肠杆菌和SF 21昆虫细胞表达和共表达隐花色素和相互作用的时钟组件。纯化的果蝇和小鼠隐花色素单独或与相互作用的dTim和mPer片段的复合物将用于通过X射线晶体学进行结晶和结构测定。光化学研究（ESR，振动和超快光谱）和时间分辨晶体学将在该联盟的合作小组中进行（R。Bittl，J. Heberle，A. Penzkarty，I.Schlichting）.与A合作。Batschauer，我们将继续进行拟南芥隐花色素At-Cry 2和At-Cry 3的结晶和晶体结构测定。

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.