Time-resolved spectroscopic characterization of blue-light photo-receptors

我们分析了衣藻蓝光受体Phot1的LOV1结构域的结构和光化学过程。我们建议分析LOV1(FMN型)三重态的电子转移过程，并研究Phot1中信号态的后续形成。我们将采取一种结合分子生物学、生物化学、光谱学、结晶学和理论的高度交叉的方法。第二，我们计划研究含有所谓的BLUF结构域的感官蓝光受体作为发色团(FAD-型)。我们将在紫色细菌R.sphaeroides中表达参与光合蛋白基因调控的Appa-蛋白，以及在鞭毛虫E.gracilis中表达作为光调控的腺苷环化酶的Pac-a/b。我们将对光激活的酶活性进行表征，并尝试确定这两种蛋白质在暗形态和光激活状态下的结构。我们将通过时间和波长分辨的UV-Vis和荧光光谱来研究主要的光过程，特别是在ns时间范围内。光谱和动力学信息将与结构数据相结合，详细描述信号态形成过程中涉及的物理和光化学反应。

We have analysed the structure and the photochemical processes of the LOV1-domain of the blue-light receptor Phot1 from Chlamydomonas. We propose to analyze the electron transfer process that has been assumed to occur in the triplet state of LOV1 (FMN-type) and to study the subsequent formation of the signaling states in Phot1.A highly interdisciplinary approach combining molecular biology, biochemistry, spectroscopy, crystallography, and theory will be undertaken. Second, we plan to study sensory blue light receptors containing the so-called BLUF domain as chromophores (FAD-type). We will express the AppA-Protein, which is involved in the regulation of genes for photosynthetic proteins in the purple bacterium R.sphaeroides, and PAC-a/b that function as light-regulated adenylate cyclases in the flagellate E.gracilis. We will characterize the light activated enzymatic activities and try to determine the structures of both proteins in the dark form, and the light-activated state. We will study the primary photoprocesses by time and wavelength resolved UV-Vis and fluorescence spectroscopy, in particular in the ns time range. The spectral and kinetic information will be combined with the structural data to describe in detail the physical and photochemical reactions involved in the formation of the signaling states.