Functional characterization of novel rhodopsins of Chlamydomonas and other algae

在目前的供资期间，我们能够确定一些迄今为止尚未描述的绿藻视紫红质的特征。我们投入的大部分精力都集中在视紫红质结构域的表征上，它是我们最近在衣藻属中发现的一个家族--组氨酸-激酶型视紫红质(HKR)的一部分。该蛋白是一种长波紫外线受体(λmax=380 nm)。紫外光吸收后，转化为稳定的偏晶型Rh-Bl(λmax=490 nm)，反应被蓝光逆转。用时间分辨UV/Vis和共振拉曼光谱研究了生色团动力学和光化学。同时，我们还研究了耐盐藻类杜氏盐藻(Dunaliella Salina)的通道视紫红质(Channel视紫质)和普通小球藻的光驱动质子泵。小球藻蛋白在光遗传方面具有很高的应用价值。在我们申请的资助期内，我们希望继续在体外和体内分子水平上对HKRs进行表征。我们将致力于在功能上表达衣藻HKR2和HKR4的视紫红质蛋白，以及研究Ostreorics的唯一HKR的光谱性质和光反应。我们将尝试表达全长蛋白，并对信号转导过程进行生化表征，包括光调节的循环酶活性。我们将通过同源重组来删除基因，并对缺失的菌株进行生物物理和生理方法的鉴定。作为一个附带项目，我们将继续对嗜冷藻Fragilariopsis lindrus和绿藻普通小球藻的唯一视紫红质进行表征。

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.