Evolution-inspired design of multicolored photoswitches from a single cyanobacteriochrome scaffold

Cyanobacteriochromes (CBCRs) are small, bistable linear tetrapyrrole (bilin)-binding light sensors which are typically found as modular components in multidomain cyanobacterial signaling proteins. The CBCR family has been categorized into many lineages that roughly correlate with their spectral diversity, but CBCRs possessing a conserved DXCF motif are found in multiple lineages. DXCF CBCRs typically possess two conserved Cys residues: a first Cys that remains ligated to the bilin chromophore and a second Cys found in the DXCF motif. The second Cys often forms a second thioether linkage, providing a mechanism to sense blue and violet light. DXCF CBCRs have been described with blue/green, blue/orange, blue/teal, and green/teal photocycles, and the molecular basis for some of this spectral diversity has been well established. We here characterize AM1-1499g1, an atypical DXCF CBCR that lacks the second cysteine residue and exhibits an orange/green photocycle. Based on prior studies of CBCR spectral tuning, we have successfully engineered seven AM1-1499g1 variants that exhibit robust yellow/teal, green/teal, blue/teal, orange/yellow, yellow/green, green/green, and blue/green photocycles. The remarkable spectral diversity generated by modification of a single CBCR provides a good template for multiplexing synthetic photobiology systems within the same cellular context, thereby bypassing the timeconsuming empirical optimization process needed for multiple probes with different protein scaffolds.