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

Abstract

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 time-consuming empirical optimization process needed for multiple probes with different protein scaffolds.

Keywords: circular dichroism; optogenetics; phytochrome.

Fig. 1.

Photochemical diversity of CBCRs. Crystal structures of DXCF CBCR TePixJ in (A) the ^15ZPb form (19) (PDB ID 4GLQ; amino acid residues are shown in cyan, and chromophore is shown in gray) and (B) the ^15EPg form (20) (PDB ID 4GLQ; amino acid residues are shown in lime green, and chromophore is shown in gray). Key mutation sites Val473, Cys494 of the second Cys, His523, Leu527, Leu530, and Asn535 in TePixJg (Te) are shown, corresponding to residues Phe97, Ser118, His147, Tyr151, Phe154, and Thr159 in AM1_1499g1 (AM). These amino acid residues are highlighted in a sequence shown below. (C) Sequence comparison between AM1_1499g1 (orange), green/teal (yellow green), blue/teal (blue), and blue/green (violet) CBCRs. Predicted secondary structures of the ^15ZPb (cyan) and the ^15EPg (lime green) forms and amino acid residues within 6 Å of the chromophores (asterisks) are based on the structures of the ^15ZPb form of TePixJg (PDB ID 4GLQ) and the ^15EPg form of TePixJg (PDB ID 3VV4). Amino acid residues of AM1_1499g1 mutated in this study (shown in sky blue) were substituted with residues found in other CBCRs (shown in orange). Highly conserved residues shown in black boldface type include the nearly invariant first Cys and the second Cys found in Asp-Xaa-Cys-Phe (DXCF) motif. (D) Phylogenetic tree of selected CBCRs and phytochromes, based on the alignment shown in SI Appendix and Dataset S1. Each lineage cluster is classified according to photocycle. CBCR subfamilies possessing the DXCF motif are indicated with asterisks.

Fig. 2.

Photocycles of AM1_1499g1 variants. (A–H) Normalized absorption spectra of AM1_1499g1 and variants are depicted with structures for each chromophore. The π-conjugated systems for each bilin are color-coded by dark-state and photoproduct-state peak absorption wavelength. (A) Wild-type AM1_1499g1 incorporates PCB and exhibits an orange/green photocycle at low (5 °C) temperature. (B) The S₁₁₈C variant incorporates PVB and exhibits a yellow/teal photocycle. (C) The S₁₁₈C/Y₁₅₁L/T₁₅₉N variant incorporates PVB and exhibits a green/teal photocycle. (D) The S₁₁₈C/H₁₄₇Y variant incorporates PVB and exhibits a blue/teal photocycle. (E) The F₉₇V variant incorporates PCB and exhibits an orange/yellow photocycle. (F) The F₉₇V/S₁₁₈C variant incorporates PVB and exhibits a yellow/green photocycle. (G) The F₉₇V/S₁₁₈C/Y₁₅₁L/T₁₅₉N variant incorporates PVB and exhibits a green/green photocycle. (H) The F₉₇V/S₁₁₈C/H₁₄₇Y variant incorporates PVB and exhibits a blue/green photocycle. Absorption maxima are reported in Table 1. Some samples, especially the F₉₇V/S₁₁₈C/H₁₄₇Y variant, showed higher absorption in the shorter-wavelength region due to scattering, indicating that these samples are unstable in solution.

Fig. 3.

Comparative photochemical difference spectra of wild-type AM1-1499g1 and its variants. Normalized difference spectra (15Z dark state – 15E photoproduct state) are shown for variants with (A) twisted or (B) relaxed D-ring photoproducts. (A) Wild-type AM1_1499g1 (^15ZPo – ^15EPg; orange), S₁₁₈C (^15ZPy – ^15EPt; yellow), S₁₁₈C/Y₁₅₁L/T₁₅₉N (^15ZPg – ^15EPt; yellow-green), and S₁₁₈C/H₁₄₇Y (^15ZPb – ^15EPt; blue). (B) F₉₇V (^15ZPo – ^15EPy; orange), F₉₇V/S₁₁₈C (^15ZPy – ^15EPg; yellow), F₉₇V/S₁₁₈C/Y₁₅₁L/T₁₅₉N (^15ZPg – ^15EPg; yellow-green), and F₉₇V/S₁₁₈C/H₁₄₇Y (^15ZPb – ^15EPg; blue). Absorption maxima are reported in Table 1.

Fig. 4.

Light-dependent adenylate cyclase activities of both photostates of AM1_1499g1_S₁₁₈C-AC. The enzymatic reaction catalyzing cAMP synthesis from ATP by the AM1_1499g1_S₁₁₈C–adenylate cyclase chimeric protein was examined after 0, 5, 10, 20, 30, and 60 min at 25 °C for ^15ZPy (yellow) and ^15EPt (teal). Reaction products were quantified using HPLC (SI Appendix, Fig. S9D and Table S3). Data are reported as mean ± SD, calculated from three independent experiments.