Zinc-Induced Fluorescence Turn-on in Native and Mutant Phycoerythrobilin-Binding Orange Fluorescent Proteins

Cyanobacteriochrome (CBCR)-derived fluorescent proteins are a class of reporters that can bind bilin cofactors and fluoresce across the ultraviolet to near-infrared spectrum. Derived from phytochrome-related photoreceptor proteins in cyanobacteria, many of these proteins use a single small GAF domain to autocatalytically bind a bilin and fluoresce. The second GAF domain of All1280 from Nostoc sp. PCC7120 is a DXCF motif-containing protein that exhibits blue light-responsive photochemistry when bound to its native cofactor, phycocyanobilin. GAF2 can also bind non-photoswitching phycoerythrobilin (PEB), resulting in a highly fluorescent protein. Given the small size, high quantum yield, and that, unlike green fluorescent proteins, bilin-binding proteins can be used in anaerobic organisms, the orange fluorescent GAF2-PEB protein is a promising platform for designing new genetically encoded metal ion sensors. Here we show that GAF2-PEB undergoes a ∼5-fold reversible zinc-induced fluorescence enhancement with blue-shifted emission maximum (572 to 517 nm), which is not observed for a related PEB-bound GAF from Synechocystis sp. PCC6803 (Slr1393g3). Zn ²⁺ significantly enhances GAF2-PEB fluorescence across a biologically relevant pH range from 6.0-9.0 and with pH-dependent µM to nM dissociation constants. Site-directed mutants aiming to sterically decrease and increase access to PEB show a decreased and similar amount of zinc-induced fluorescence enhancement, respectively. Mutation of the cysteine residue within the DXCF motif to alanine abolishes zinc-induced fluorescence enhancement. Collectively, these results support the presence of a fluorescence enhancing Zn ²⁺ binding site in GAF2-PEB likely involving coordination to the bilin cofactor and requiring a nearby cysteine residue.