A Single‐Site Mutation Tunes Fluorescence and Chromophorylation of an Orange Fluorescent Cyanobacteriochrome**

Abstract

Cyanobacteriochrome (CBCR) cGMP‐specific phosphodiesterase, adenylyl cyclase, and FhlA (GAF) domains bind bilin cofactors to confer sensory wavelengths important for various cyanobacterial photosensory processes. Many isolated GAF domains autocatalytically bind bilins, including the third GAF domain of CBCR Slr1393 from Synechocystis sp. PCC6803, which binds phycoerythrobilin (PEB) to yield a bright orange fluorescent protein. Compared to green fluorescent proteins, the smaller size and lack of an oxygen requirement for fluorescence make Slr1393g3 a promising platform for new genetically encoded fluorescent tools. Slr1393g3, however, shows low PEB binding efficiency (chromophorylation) at ~3% compared to total Slr1393g3 expressed in E. coli. Here we used site‐directed mutagenesis and plasmid redesign methods to improve Slr1393g3‐PEB binding and demonstrate its utility as a fluorescent marker in live cells. Mutation at a single site, Trp496, tuned the emission over ~30 nm, likely by shifting autoisomerization of PEB to phycourobilin (PUB). Plasmid modifications for tuning relative expression of Slr1393g3 and PEB synthesis enzymes also improved chromophorylation and moving from a dual to single plasmid system facilitated exploration of a range of mutants via site saturation mutagenesis and sequence truncation. Collectively, the PEB/PUB chromophorylation was raised up to a total of 23% with combined sequence truncation and W496H mutation.

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

“…The spectroscopic properties of All1280g2-PEB/PUB and Slr1393g3-PEB/PUB were similar to those previously reported (Figures B,C and S4, Table S2). , The spectroscopic properties of the All1280g2-PEB mutants are discussed below (Figure S4).…”

“…(A) General chemical structure of PEB in solution and upon reaction with a GAF protein cysteine residue. Partial autoisomerization of GAF-PEB to phycourobilin (PUB) can occur upon bilin binding. , (B) UV–visible and (C) fluorescence excitation (λ em = 620 nm) and emission (λ ex = 495 nm) spectra of purified All1280g2-PEB/PUB [10 μM in (B) and 3 μM in (C)]. Buffer: 50 mM HEPES, 100 mM NaCl, pH 7.1.…”

“…The plasmids pACYC-PebS-HO1 and pET28a-Slr1393g3 were reported previously . The plasmid pACYC-PebS-HO1 encodes the heme oxygenase gene (HO1, UniProtKB entry P72849) from pSR43.6r (Addgene no.…”

“…Similar proteins from our group and others have, however, been re-engineered to improve the binding efficiency of alternative bilin cofactors, suggesting that similar improvements may be achieved in All1280g2. 11,53,67 Finally, we note that while the zinc-induced fluorescence response is reversible up to pH ∼ 7.5, the reversibility gradually decreases as the pH is raised further; therefore, mutations that alter the binding site pK a will need to be carefully considered. Future work will aim to better understand and predict the effects of the bilin binding pocket amino acid residues on zinc-induced fluorescence enhancement and metal coordination within the bilin binding site.…”

“…The plasmids pACYC-PebS-HO1 and pET28a-Slr1393g3 were reported previously. 53 The plasmid pACYC-PebS-HO1 encodes the heme oxygenase gene (HO1, UniProtKB entry P72849) from pSR43.6r (Addgene no. 63197, a gift from Jeffrey Tabor) 54 and the PebS gene from Prochlorococcus phage P-SSM2 (UniProtKB entry Q58MU6, codon optimized for E. coli).…”