Optogenetics for all: A general method for optical control of protein activity

描述(申请人提供)：对基因定义的蛋白质活动的光学控制，即蛋白质的光遗传调节，长期以来一直是生物学中的梦想。如果我们能够开发一种通用的方法来光学控制感兴趣的蛋白质的活性，它可以深刻地改变生物实验，并赋予基于基因和细胞的治疗新的能力。例如，蛋白质协调的细胞功能，如生存、凋亡、分化、迁移或连接(在神经系统中)可以在体内进行控制，以微米级的分辨率研究生物生理学。治疗性植入的细胞可以类似地受光控制，以精确地将治疗集中在所需的解剖位置。还有无数其他可能的应用。鉴于光学蛋白调控的这些优势，人们已经花费了相当大的努力来调整已知的光响应信号结构域来调控哺乳动物蛋白质。然而，现有的策略需要广泛的筛选来创造光响应蛋白，或者依赖于蛋白质的重新定位来间接调节活性。因此，这些方法只被用来控制少数蛋白质。因此，需要一种方法来产生光调节的感兴趣蛋白质， 是可以概括的。我们最近发现了一类新的光介导的蛋白质-蛋白质相互作用，并将这一发现转化为一种可推广的用光控制蛋白质活动的方法。我们假设荧光蛋白(FP)可以经历光依赖的构象变化，从而驱动寡聚化状态的变化。事实上，我们发现可逆光开关绿色FP Dronpa的四聚体和二聚体变体在被青色光从明亮状态切换到黑暗状态时经历解离。然后我们发现，融合在感兴趣的酶结构域两端的Dronpa结构域将其关在黑暗中，但允许在Illumina上取消连接

DESCRIPTION (provided by applicant): Optical control of genetically defined protein activities, i.e. optogenetic regulation of proteins, has long been a dream in biology. If we could develop a generalizable method to optically control activities of proteins of interest, it could profoundly transform biological experimentation and impart new capabilities to gene- and cell-based therapies. For instance, the cellular functions that proteins coordinate, such as survival, apoptosis, differentiation, migration or connectivity (in the nervous system) could be controlled in vivo to study organismal physiology with micron-level resolution. Therapeutically implanted cells could be similarly controlled by light to precisely focus treatment at desired anatomical locations. There are countless other possible applications. Given these advantages of optical protein regulation, considerable efforts have been expended to adapt known light-responsive signaling domains to regulate mammalian proteins. However, existing strategies require extensive screening to create light-responsive proteins, or rely on protein relocalization to indirectly regulate activity. As a result, these methods have been used to control only a few proteins. Thus there exists a need for a method to create light-regulated proteins of interest that is generalizable. We have recently discovered a new class of light-mediated protein-protein interaction, and translated this discovery into a generalizable method for controlling protein activities with light. We hypothesized that fluorescent proteins (FPs) could undergo light-dependent conformational changes that drive changes in oligomerization state. Indeed, we found that tetrameric and dimeric variants of the reversibly photoswitching green FP Dronpa undergo dissociation as they are switched from bright to dark states by cyan light. We then discovered that fusing Dronpa domains at both ends of an enzymatic domain of interest cages it in the dark but allows uncaging upon illumina