Light responses of animal-like cryptochromes and aureochromes from microalgae

隐色素和金黄色色素是植物的光感受器，含有黄素作为发色团。这些受体在微藻中的作用机制尚不清楚。隐色素在高等植物中调控蓝光依赖性过程，如光形态形成和昼夜节律。然而，在莱茵衣藻（Chlamydomonas reinhardtii）中，发现了另一种类似动物的隐色素（aCRY）。aCRY是目前唯一一种不仅能被蓝光激活，还能被红光激活的感觉黄蛋白。这种独特的aCRY红光敏感性的结构基础需要阐明。我们已经证明异种表达的aCRY形成黄素的中性自由基，其吸收光谱与体内的光谱响应相似。因此，中性自由基态被假定为暗态。还原剂和点突变对暗态形成和稳定的影响将被研究。光化学反应的过程将用时间分辨光谱学来记录。aCRY不同于同源蛋白，其功能是作为核心时钟成分或特定c端延伸的DNA修复酶。红外差分光谱学将应用于搜索光依赖的结构变化在这个扩展。进一步有希望的候选微藻感觉隐色素将被光谱表征。到目前为止，只在藻类中发现了金黄色色素，包括硅藻褐毛藻。金色素不同于所有其他含有LOV（光、氧或电压敏感）结构域的蛋白质，如趋光蛋白，其效应域和传感器域的反向排列。我们已经证明，尽管排列相反，c端LOV-J（α）螺旋在金黄色色素中展开，与趋光素相似。我们将利用红外双差光谱研究全长AUREO1a中J（α）螺旋或其他片段的信号传递进展。该转录因子的DNA结合能力将被研究。其他的金黄色色素将异种表达，并分析其光化学性质及其在体内的作用。对这些光致变色和隐致变色信号传递新途径的研究将大大拓宽我们目前对光感器的理解。

Cryptochromes and aureochromes are plant photosensors incorporating a flavin as a chromophore. The mechanism and the role of these receptors in microalgae remain largely unclear. Cryptochromes regulate blue-light-dependent processes such as photomorphogenesis and circadian rhythm in higher plants. In the green alga Chlamydomonas reinhardtii, however, an additional animal-like cryptochrome (aCRY) is found. aCRY is currently the only sensory flavoprotein, which is activated not only by blue but also by red light. The structural basis of this unique red-light sensitivity of aCRY needs to be elucidated. We have demonstrated that heterologously expressed aCRY forms a neutral radical of flavin with an absorption spectrum similar to the spectral response in vivo. The neutral radical state was therefore postulated as dark state. The influence of reductants and point mutations on formation and stabilisation of this dark state will be investigated. The course of the photoreaction will be followed by time-resolved spectroscopy. aCRY differs from homologous proteins with a function as core clock component or DNA repair enzyme in a specific C-terminal extension. Infrared difference spectroscopy will be applied to search for lightdependent structural changes in this extension. Further promising candidates for sensory cryptochromes from microalgae will be characterized by spectroscopy. Aureochromes have only been identified in algae up to now including the diatom Phaeodactylum tricornutum. Aureochrome differs from all other proteins containing LOV (light-, oxygen-, or voltage-sensitive) domains such as phototropin in a reverse arrangement of effector and sensor domains. We have shown that despite the reverse arrangement, the C-terminal LOV-J(alpha) helix unfolds in aureochrome similar to phototropin. We will investigate the progress of signal transfer by the J(alpha) helix or other segments in the full-length AUREO1a using infrared double difference spectroscopy. The DNA binding ability of the putative transcription factor will be studied. Other aureochromes from P. tricornutum will be heterologously expressed and their photochemical properties will be analyzed with respect to their role in vivo. The investigation of the novel pathways of signal transfer in these aureochromes and cryptochromes will significantly broaden our current understanding of photosensors.