Red-light-only control of protein-protein interactions using a cyanobacteriochrome (UNICYCL)

Most optogenetic tools are controlled by blue light. Red-light responsive tools enable multiwavelength applications and allow greater biological tissue penetration with reduced toxicity. Current red-light tools are primarily based on phytochromes, large dimeric proteins with a structurally complex mode of interaction with their binding partners. Here we introduce a small red-light-only responsive system composed of a GA domain (6 kDa) that binds to a CBCR GAF domain (17 kDa) with a K _d ∼1-5 µM to form a 1:1 complex in the dark. Red light causes dissociation of the complex by causing a >25-fold decrease in binding affinity. The CBCR GAF domain reverts to the dark state with a half-life of ∼1 min and the complex reforms. Structural analysis using NMR measurements combined with molecular docking and dynamics simulations shows that the binder interacts with the GAF domain and senses isomerization of the bilin chromophore at a site that overlaps the critical tongue domain of phytochromes. This system provides a small, simple red-light-only optogenetic tool that can operate to control protein-protein interactions in vitro and in living cells.