A novel photoreaction mechanism for the circadian blue light photoreceptor Drosophila cryptochrome
A novel photoreaction mechanism for the circadian blue light photoreceptor Drosophila cryptochrome
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DOI：
10.1074/jbc.m608872200
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发表时间：
2007-04-27
影响因子：
4.8
通讯作者：
Wolf, Eva
中科院分区：
文献类型：
--
作者：
Berndt, Alex;Kottke, Tilman;Wolf, Eva
隐花色素是一种与DNA光裂合酶进化相关的黄素蛋白，但缺乏DNA修复活性。果蝇隐花色素（dCRY）是一种蓝光光感受器，参与生物钟与环境光暗周期的同步。到目前为止，光谱和结构的研究，这和其他动物隐花色素在很大程度上阻碍了他们的重组表达的困难。因此，我们建立了一种表达和纯化方案，使我们能够从Sf 21昆虫细胞培养物中纯化mg量的单体dCRY。使用紫外-可见光谱，质谱和反相高压液相色谱，我们表明，昆虫细胞纯化的dCRY含有黄素腺嘌呤二核苷酸在其氧化态（FAD（ox））和残留量的亚甲基四氢叶酸。在蓝光照射下，dCRY经历可逆的吸收变化，这归因于FAD（ox）转化为红色阴离子FAD（-.）激进我们的发现使我们提出了一种新的dCRY光反应机制，其中FAD（ox）对应于基态，而FAD（-.）自由基代表介导果蝇昼夜节律钟重置的光激活状态。
Cryptochromes are flavoproteins that are evolutionary related to the DNA photolyases but lack DNA repair activity. Drosophila cryptochrome (dCRY) is a blue light photoreceptor that is involved in the synchronization of the circadian clock with the environmental light-dark cycle. Until now, spectroscopic and structural studies on this and other animal cryptochromes have largely been hampered by difficulties in their recombinant expression. We have therefore established an expression and purification scheme that enables us to purify mg amounts of monomeric dCRY from Sf21 insect cell cultures. Using UV-visible spectroscopy, mass spectrometry, and reversed phase high pressure liquid chromatography, we show that insect cell-purified dCRY contains flavin adenine dinucleotide in its oxidized state (FAD(ox)) and residual amounts of methenyltetrahydrofolate. Upon blue light irradiation, dCRY undergoes a reversible absorption change, which is assigned to the conversion of FAD(ox) to the red anionic FAD(-.) radical. Our findings lead us to propose a novel photoreaction mechanism for dCRY, in which FAD(ox) corresponds to the ground state, whereas the FAD(-.) radical represents the light-activated state that mediates resetting of the Drosophila circadian clock.