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

隐色素是FAD和MTHF结合蛋白。果蝇隐花色素（dCry）是一种蓝光光感受器，参与昼夜节律（24小时）与环境暗-光周期的同步。两种小鼠隐色素（mCry 1和mCry 2）以不依赖光的方式作为小鼠昼夜节律振荡器的组成部分。本课题旨在利用x射线晶体学、生物化学和光谱学技术，研究果蝇和小鼠隐色素及其与效应分子复合物的结构和光化学。我们利用大肠杆菌和SF21昆虫细胞表达和共表达隐色素和相互作用的时钟成分。纯化的果蝇和小鼠隐色素单独或与dTim和mPer片段相互作用的复合物将用于x射线晶体学的结晶和结构测定。光化学研究（ESR，振动和超快光谱）和时间分辨晶体学将在联盟的合作小组（R. Bittl， J. Heberle， A. Penzkofer， I.Schlichting）中进行。与A. Batschauer合作，我们将继续进行A.thaliana Cryptochromes At-Cry2和At-Cry3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.