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

天然光感受器的分析和改造，以光操纵动物细胞中的环核苷酸、Ca2 和膜电压

• 批准号: 164476362
• 负责人: Professor Dr. Georg Nagel
• 金额: --
• 依托单位: Lehrstuhl für Physiologie II - Neurophysiologie
• 依托单位国家: 德国
• 项目类别: Research Units
• 财政年份: 2010
• 资助国家: 德国
• 起止时间: 2009-12-31 至 2013-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/164476362?language=en
• 关键词: Analysis; engineering; natural; photoreceptors; Light

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.

来自鞭毛藻Euglena gracilis的黄素蛋白-光活化腺苷酸环化酶（PAC）的克隆启动了一种新的光遗传学工具的使用，因为纯化的PACα2β2显示了cAMP的光诱导产生。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[cAMP]增加，尽管PACα的效果要高出100倍。本研究拟通过对PACα和PACβ进行突变，研究PACα和PACβ的相互作用以及重要氨基酸的功能。最近，我们通过突变PACα的实验表明，它作为PACα2二聚体发挥作用。我们将与致力于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转化为光活化的鸟苷酸环化酶（PGC），这是我们研究小组合作者与模式生物合作的新工具（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点，并在功能测试后将其提供给我们的合作者。我们建议表达微生物型视紫红质（光激活的Cl-泵halorhodopsin和光门控阳离子通道channelrhodopsin-2）作为片段，分裂成两个蛋白质部分，并在膜中测试功能重建。在两种不同启动子的控制下，这样的构建体将使更多的细胞特异性表达成为可能，特别是在果蝇的神经系统中。线虫和斑马鱼我们将荧光蛋白（FP）插入到视紫红质的螺旋间环中，以确认这些环，并增强从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，在该项目中，利用表面增强光谱技术研究了视紫红质片段在支撑膜上的相互作用。