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 illumination. Remarkably, this simple strategy was capable of controlling multiple types of domains. Furthermore, we could use off-switching of Dronpa fluorescence to track the extent of protein uncaging. Thus our strategy is generalizable and conveniently uses the same FP domain to confer light control and report on a proteins activity. We believe FLIPs could be the long-awaited method for controlling biological pathways by light easily and robustly. While we have established initial proof of concept, we still have questions about how optical control of FLIPs occurs at the atomic level, whether the FLIP approach can be extended to other wavelengths and to proteins of various topologies, and whether optical regulation of FLIPs can successfully control biology in a non-toxic manner in living animals. By addressing these important questions, we will develop a robust toolkit and guide for creating optically controlled proteins that can be used by all biologists, thereby converting optical control of biology from dream to reality.
Studies on how the activities of proteins cause disease or can ameliorate it, and the development of effective cell-based therapies for regenerative medicine, would benefit enormously from a way to control specific protein activities precisely in space and time. Light is the ideal method for controlling protein activities as it can be rapidly and inexpensively modulated. In the proposed work, we will apply our recent discovery of a new light-controlled protein-protein interaction to develop and test a general approach to controlling any protein of interest with light.
