Conversion of phycocyanobilin-binding GAF domain to biliverdin-binding domain

Abstract

Cyanobacteriochromes (CBCRs) are biliprotein photoreceptors that only exist in cyanobacteria and have a broad spectral response range from ultra-violet to far-red. The red/green-type CBCRs can show red/green reversible photoconversion via a covalently bound phycocyanobilin (PCB). In recent years, several CBCRs binding with not only PCB but also biliverdin (BV) have been discovered, which raises the possibility of CBCRs being applied as optogenetic tools. Through molecular modification, we hope to engineer BV-binding CBCRs responsive to the near-infrared spectral region (650–900 nm), of which the red/green type of CBCRs are suitable resources for experimentation. Here, we use Slr1393g3 (the third GAF domain of a red/green photoswitching CBCR from Synechocystis sp. PCC 6803) as a template to perform such molecular evolution using both random mutagenesis and site-directed mutagenesis. After several rounds of random mutagenesis, we obtained several BV-binding variants of Slr1393g3. These BV-binding variants have a maximal absorbance at ̃690 nm and a fluorescence at ̃720 nm. Additionally, some of them have remarkable photochromicity between a far-red light-absorbing state and a red light-absorbing state. Based on the primary amino acid sequence and structural models, the Phe474 surrounding ring D of BV is thought as a crucial site for chromophore selectivity.

Computational identification of key residues regulating fluorescence emission in a red/green cyanobacteriochrome

“…We first identified residues that can be targeted for regulating red‐shifted fluorescence wavelength by inspecting residue‐wise mutational perturbation on protein stability. We examined 28 variants of BV‐binding Slr1393g3 with different Pfr fluorescence wavelengths 30 . Each variant has 4–13 multiple random mutations, and the fluorescence emission peak ranges from 685 to 732 nm.…”

“…We hypothesized that the stability enhancement of the α3 region (along with L495H and Q497H mutations) contributes to the red‐shifted fluorescence wavelength. To validate the hypothesis further, we chose V‐4‐1 724 as a reference structure to generate new variants using Rosetta because V‐4‐1 724 showed better BV‐binding and have both L495H and Q497H mutations 30 . In the α3 helix, five residue positions, 469, 499, 502, 504, and 505 (WAS index >0.5) are selected for sequence design (Figure 6).…”

“…To study the key factors for spectral variation in BV‐binding Slr1393g3, 30 variants that exhibited photoconversion between Pfr and Pr states generated using random mutagenesis were collected from the literature 30 . From multiple sequence alignment (MSA), two variants that are identical to other variants are removed (Figure S1 and S2).…”

“…PCC6803 consists of three consecutive GAF domains, out of which only the third GAF domain (Slr1393g3) binds to PCB and shows reversible red/green photoconversion 8,14,26–29 . In a recent study, multiple CBCR variants from Slr1393g3 capable of solely binding to BV were generated by molecular evolution, and some of them displayed phenomenal far‐red/red (Pfr/Pr) photoconversion 30 . In the study, various BV‐binding CBCR variants exhibiting different fluorescence spectra were generated by random mutagenesis.…”

“…8,14,[26][27][28][29] In a recent study, multiple CBCR variants from Slr1393g3 capable of solely binding to BV were generated by molecular evolution, and some of them displayed phenomenal farred/red (Pfr/Pr) photoconversion. 30 In the study, various BV-binding CBCR variants exhibiting different fluorescence spectra were generated by random mutagenesis. Combined with the crystal structure data, 28 these multiple variants have the potential to provide crucial information for identifying the residues that govern the fluorescent properties of CBCRs.…”