Analysis and engineering of natural photoreceptors to Light-manipulate cyclic nucleotides, Ca2+ and membrane voltage in animal cells

从纤细鞭毛虫(Euglena Gracilis)中克隆出一种黄素蛋白--光活化腺酰环化酶(PAC)，这开启了一种新的光遗传工具的使用，因为纯化的PACα2β2光诱导产生cAMP。我们在动物细胞中分别表达了pacα和pacβ，并显示出快速和可逆的光诱导的[cAMP]升高，尽管pacα的效果是两者的100倍。我们打算通过突变Pacα和Pacβ来研究重要氨基酸的功能以及Pacα与Pacβ的相互作用。最近，我们通过对突变的pacα的实验表明，它具有pacα2二聚体的功能。我们将与研究蓝光受体结构的合作者密切合作(项目6)。我们的目标是将PAC转化为光活化鸟苷酸环酶(PGC)，这是我们研究小组中的合作者与模型生物合作的新工具(项目1、5、7)。我们计划通过将PAC与不同的膜蛋白融合，将其定位于特定的膜位置，并在功能测试后将其提供给我们的合作者。我们建议将微生物类型的视紫红质(光激活的氯泵卤视紫质和光门控阳离子通道视紫红质-2)作为片段表达，分成两个蛋白质部分，并测试膜上的功能重建。这种结构在两个不同启动子的控制下，将使更多的细胞特异性表达，特别是在果蝇、线虫和斑马鱼的神经系统中。我们将把荧光蛋白(FP)插入到视紫红质的螺旋间环中，以确认这些环，并促进从FP中间分裂的两个片段重建功能性视紫红质。我们将与项目2合作，利用表面增强光谱技术研究视紫红质片段在支撑膜上的相互作用。

The cloning of a flavoprotein from the flagellate Euglena gracilis – Photoactivated Adenylyl Cyclase (PAC) – initiated the use of a new optogenetic tool, as light-induced production of cAMP by purified PACα2β2 was shown. We expressed PACα and PACβ separately in animal cells and showed fast and reversible light-induced [cAMP] increase with both, although 100-fold more effective with PACα. We intend to mutate PACα and PACβ to study the function of important amino acids and the interaction of PACα with PACβ. Recently we showed by experiments with mutated PACα that it functions as a PACα2 dimer. We will closely collaborate with collaborators working on the structure of blue-light receptors (project 6). We aim to convert PAC into a photo-activated guanylyl cyclase (PGC), a new tool for our collaborators in the research group, working with model organisms (projects 1, 5, 7). We plan to localize PAC to specific membrane sites by fusing it to different membrane proteins and make it available – after functional tests – to our collaborators. We propose to express microbial type rhodopsins (the light-activated Cl- pump halorhodopsin and the light-gated cation channel channelrhodopsin-2) as fragments, split into two protein moieties and to test functional reconstitution in the membrane. Such constructs, under the control of two different promoters, will enable more cellspecific expression, especially in the nervous system of Drosophila, C. elegans, and zebrafish. We will insert fluorescent proteins (FP) into interhelical loops of rhodopsins to confirm these loops and to enhance reconstitution of a functional rhodopsin from two fragments, split in the middle of the FP. We will collaborate with project 2 where the interaction of rhodopsin fragments is studied on supported membranes with surfaceenhanced spectroscopic techniques.