{"doi":"10.1101/2023.08.11.552977","title":"Zinc-Induced Fluorescence Turn-on in Native and Mutant Phycoerythrobilin-Binding Orange Fluorescent Proteins","abstract":"Abstract 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.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2023,"id":410296,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9605,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2023-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1150758,"name":"Makena K. Janis","orcid":"0000-0002-2149-7410","position":1,"is_corresponding":false},{"id":1151115,"name":"J. R. Jara","orcid":null,"position":2,"is_corresponding":false},{"id":1151116,"name":"Nasir Abbasi","orcid":null,"position":3,"is_corresponding":false},{"id":855593,"name":"Melissa L. Zastrow","orcid":null,"position":4,"is_corresponding":false},{"id":1150757,"name":"Gary C. Jensen","orcid":"0000-0001-8215-2590","position":0,"is_corresponding":true}],"reference_count":56,"raw_metadata":null,"created_at":"2026-07-19T01:21:35.071397Z","pmid":"37609204","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}