Functional characterization of novel rhodopsins of Chlamydomonas and other algae

在目前的资助期间，我们能够表征一些迄今为止未描述的绿色藻类的视紫红质。我们已经投入了大部分的努力在视紫红质结构域的表征，这是一个新的组氨酸激酶视紫红质（HKR），我们最近在衣原体发现的家庭的一部分。该蛋白是UVA-受体（λmax = 380 nm）。在紫外光吸收后，其转化为稳定的中间型Rh-Bl（λmax = 490 nm），并且该反应被蓝光逆转。通过时间分辨紫外/维斯光谱和共振拉曼光谱研究了发色团动力学和光化学。同时，我们的特点是一个嗜盐杜氏盐藻，专门进行H+，和光驱动的质子泵的小球藻。小球藻蛋白对于光遗传学应用具有高度兴趣。在我们申请的资助期间，我们希望继续在体外和体内分子水平上对HKR进行表征。我们将致力于功能性表达衣原体的HKR 2和HKR 4以及Ostreococcus的唯一HKR的视紫红质，以研究光谱特性和光反应。我们将尝试表达全长蛋白质和生化表征的信号转导过程，包括光调节环化酶活性。我们将通过同源重组删除基因，并通过生物物理和生理方法对删除菌株进行表征。作为一个附带项目，我们将继续对嗜冷的柱状拟脆藻和绿色小球藻中仅有的视紫红质进行表征。

During the current funding period we were able to characterize a number of so far non described rhodopsins of green algae. Most efforts we have invested in the characterization of rhodopsin domain that is part of the novel Histidin-Kinase Rhodopsin (HKR), a family that we recently discovered in Chlamydomonas. This protein is an UVA-receptor (λmax = 380 nm). After UV-light absorption it is converted into the stable Metaform Rh-Bl (λmax = 490 nm) and the reaction is reversed by blue light. Chromophore dynamics and photochemistry were studied by time resolved UV/Vis and Resonance Raman spectroscopy. In parallel we have characterized a Channelrhodopsin of the halotolerant alga Dunaliella salina, which exclusively conducts H+, and a light driven proton pump of Chlorella vulgaris. The Chlorella protein is of high interest for optogenetic applications. During the funding period we are applying for we would like to continue the characterization of the HKRs on a molecular level in vitro and in vivo. We will undertake efforts to functionally express the rhodopsins of HKR2 and HKR4 of Chlamydomonas and the only HKR of Ostreococcus to study spectral properties and photoreactions. We will try to express the full-length proteins and to biochemically characterize the signaling process including light regulated cyclase activity. We will delete the genes by homologous recombination and characterize the deletion strains by biophysical and physiological methods. As a side project we will continue the characterization of the only rhodopsins of the cryophilic alga Fragilariopsis cylindrus and the green alga Chlorella vulgaris.