Phylogenetic tree, absorption spectra, and light-induced Ca2+ responses of Acropora tenuis opsins belonging to the ASO-II group.

(A) Maximum-likelihood (ML) tree of animal opsins including A. tenuis opsins in the ASO-II group. Seven opsins in the ASO-II group that were identified and cloned from A. tenuis in this study are shown in bold and the three members for which we obtained absorption spectra are highlighted in red. Numbers at the nodes represent support values of each ML branch estimated by 1000 bootstrap samplings (≥ 70% are indicated). Scale bar = 0.6 substitutions per site. All branches and support values are provided in Fig. S1. (B) Absorption spectra in the dark of three A. tenuis opsins in the ASO-II group (Antho2a, Antho2c and Antho2e). The absorption spectra were measured at 0°C in 140 mM NaCl at pH 6.5. The number in each graph shows the λmax value. (C) Results of the aequorin-based bioluminescent reporter assay for monitoring light-induced changes in Ca2+ in HEK293S cells expressing the same three opsins in the ASO-II group as in B. In each graph, luminescence values were normalised to the baseline. Black circles with error bars indicate the means ± SEMs (n = 3) of the measured relative luminescence. Black arrowheads at time 0 indicate the timing of one-minute irradiation with green (495 nm; for Antho2a and Antho2c) or UV (395 nm; for Antho2e) light.

Absorption spectra of wild type and the E292A mutant of A. tenuis Antho2a.

(A, B) Absorption spectra of the dark state (curve 1, black) and the photoproduct (curve 2, red) of the wild type (Antho2a WT, A) and the E292A mutant (Antho2a E292A, B) at 140 mM NaCl and pH 6.5. The samples were kept at 0°C during the spectroscopic measurements. (C, D) Absorption spectra of the dark state of Antho2a WT (C) and Antho2a E292A (D) prepared in Cl−-depleted conditions, before (curve 1, black) and after (curve 2, blue) adding Cl− (see Materials and Methods for details). In the Cl−-depleted condition, the pigments were solubilised in 70 mM Na2SO4, which reportedly does not access to the Cl− binding site in the chicken red-sensitive cone visual pigment iodosin (Shichida et al., 1990), to moderate protein denaturation.

Effects of pH and Cl− concentration on the absorption spectra of the dark states of wild type Antho2a and the Antho2a E292A mutant.

(A, B) Changes in the absorbance at λmax as a function of pH for (A) wild type Antho2a and (B) the E292A mutant at different Cl− concentrations. The absorbance values at “visible λmax” (mean absorbance at 503 ± 5 nm for the wild type and 505 ± 5 nm for the E292A mutant, respectively) were normalised for each Cl− concentration to those at the lowest pH, in which the Schiff base is assumed to be fully protonated (“Rel. abs. at visible λmax” in the y-axes). Solid and dashed lines represent sigmoid fits to the experimental data for each Cl− concentration (indicated by different colours). The pH-dependent change of wild type Antho2a at 140mM NaCl is also shown in B (dotted grey line). The full absorption spectra used to generate these plots are provided in Fig. S7 (for wild type Antho2a) and Fig. S9 (for the E292A mutant). (C) Changes in the absorbance at λmax for wild type Antho2a (black open circles) and the E292A mutant (red solid circles) as a function of Cl− concentration. The absorbance values at visible λmax were normalised to those at 500 mM NaCl for both the wild type and the E292A mutant. The lines in the graph were generated by fitting the Hill equation to the experimental data.

Structural models of the retinal binding pocket in the dark state of (A, B) wild type Antho2a and (C) the E292A mutant obtained using QM/MM calculations. For wild type Antho2a, two models are shown: (A) with a protonated (neutral) Glu292 and (B) a deprotonated (negatively charged) Glu292.

Comparison of the light-evoked intracellular Ca2+ levels between wild type Antho2a and the E292A mutant.

The graph shows the mean ± S.E.M (n = 4) of the measured relative luminescence values (luminescence values normalised to the baseline) for wild type Antho2a (black) and the E292A mutant (red). The green vertical line indicates the time of cell illumination with green light (510 nm, for 1 s, 1.65 × 1015 photons/cm2/s).

pH-dependent changes in the absorption spectra of Antho2c and Antho2e at different Cl− concentrations at 0°C.

(A–C) Absorption spectra of purified wild type Antho2c pigment at (A) 0 mM, (B) 0.093 mM, and (C) 9.3 mM NaCl concentrations. The corresponding pH values are indicated on each curve in the graphs. (D) Summary of the spectral changes for wild type Antho2c across different Cl− concentrations at neutral pH (pH 6.5). (E, F) Absorption spectra of (E) wild type Antho2e (Antho2e WT) and (F) its R113A mutant (Antho2e R113A) at different Cl− concentrations at pH 6.5 at 0°C. Each colour indicates a different Cl− concentration.

Vertical excitation energies (ΔEcalc) and oscillator strengths (f) computed by quantum mechanics/molecular mechanics (QM/MM) calculations using different QM methods with the cc-pVTZ basis set.

Maximum-likelihood (ML) tree of animal opsins, with non-opsin GPCRs included as an outgroup (a simplified version of the ML tree is shown in Fig. 1A). The tree includes opsins belonging to the eight main groups (see main text) as well as of opsins from the recently identified subgroups xenopsins and chaopsins6. The sixteen A. tenuis opsins (eight opsins in the cnidopsin group, one opsin in the ASO-I group, and seven opsins in the ASO-II group) that were identified in this study are shown in bold. The three opsins in the ASO-II group for which absorption spectra were successfully obtained are highlighted in red. Numbers at nodes represent support values for the ML branch estimated by 1000 bootstrap samplings (≥ 70% are indicated). Scale bar = 0.6 substitutions per site.

Amino acid sequence alignment of opsins in the ASO-II group (in red) with other animal opsins.

Only selected regions of the full alignment are shown, with their secondary structure elements displayed at the top. The alignment reveals residues and motifs in the ASO-II group that are broadly conserved throughout the large family of Class A GPCRs, such as the key functional residue D83 (grey) in TM2, a disulphide bridge between C110 in TM3 and C187 in ECL2 (black), and the NPxxY motif in TM7 (blue). Strikingly, ASO-II opsins lack the highly conserved and functionally key E(D)RY motif on TM3 (green). ASO-II opsins also possess residues specific to animal opsins, such as the retinal binding K296 (red). While they do not have E/D at any of the three established counterion positions (yellow), they feature a highly conserved. Glu/Asp at position 292 (orange).

Photo- and thermal reactions of wild type Antho2a.

(A, B) Absorption spectra and isomer compositions of the chromophore retinal of Antho2a WT before and after light irradiation. (A) Absorption spectra were measured at 0°C in the dark (curve 1, black), after orange light irradiation (> 560 nm, curve 2 and curve 3, deep and pale orange), and after subsequent violet light irradiation (420 nm, curve 4, purple). Irradiation of wild type Antho2a with orange light shifted the absorption maximum from 503 nm in the dark (curve 1, black) to 476 nm in the photoproduct (curve 2 and curve 3, orange). Upon subsequent irradiation with 420 nm light (while the photoproduct was stable), the λmax of the photoproduct stayed at 476 nm, with only a slight decrease of the peak absorbance (curve 4, purple) possibly resulting from degradation of the photoproducts upon light irradiation. (B) The configuration of retinal in Antho2a WT before (black) and after (orange) irradiation with orange light (> 560 nm) was analysed with HPLC. Retinal was extracted in its oxime form. AT, all-trans retinal; 11, 11-cis retinal. (C, D) Thermal stability of the photoproducts of wild type Antho2a in 140 mM NaCl at pH 6.5 and 0 °C. (C) Changes in the absorption spectra of Antho2a WT after irradiation with orange (>560 nm) light (with the sample kept in the dark at 0°C). Each coloured curve corresponds to a different time after light irradiation. (D) Difference spectra obtained by subtracting the spectrum of Antho2a WT in the dark from the spectra measured at different time points after irradiation (shown in C).

(A, B) Effect of pH on the absorption spectra of the dark state and photoproduct of (A) wild type Antho2a and (B) the E292A mutant at 140 mM NaCl.

Each graph shows spectra before (curves 1, black) and after (curves 2, red) irradiation with orange (> 560 nm) light.

Effect of pH and NaCl concentration on absorption spectra of the dark states and photoproducts of Antho2a.

(A) Absorption spectra of wild type Antho2a at 0.28 mM NaCl under different pH conditions (pH 6.5 and pH 7.3). (B) Absorption spectra of wild type Antho2a at pH 6.5 with varying NaCl concentrations (2.8 mM, 8 mM, and 800 mM). (C) Absorption spectra of the E292A mutant of Antho2a at 800 mM NaCl and different pH conditions (pH 4.8, pH 6.6, and pH 7.6). Each graph shows spectra before (curves 1, black) and after (curves 2, deep purple/red, and 3, pale purple/pink) irradiation with UV light (< 410 nm, A) or orange light (> 550 nm or > 560 nm, B and C). Curves 2 and 3 in each graph represent, respectively, the first measurement (immediately after irradiation) and a subsequent measurement (< 5 min. after irradiation). The minimal change in the absorption spectra over this time scale indicates that the spectra remain stable after irradiation.

Effect of halide anions on the absorption spectra of (A) wild type Antho2a and (B) the Antho2a E292A mutant at pH 6.5 and 0°C.

The graphic shows the normalised absorption spectra of the pigments prepared in 140 mM NaCl (black curves), 140 mM NaBr (blue curves), and 140 mM NaI (red curves).

pH-dependent changes in the absorption spectra of Antho2a WT at (A) 0.28 mM, (B) 2.8 mM, (C) 28 mM, (D) 140 mM, and (E) 500 mM Cl− at 0°C.

The pH values of the solution, measured right after each spectroscopic measurement, are indicated on the corresponding curves in each graph.

pH-dependent changes in the absorption spectra of Antho2a WT and Antho2a E292A at 0 mM NaCl (containing 70 mM Na2SO4) at 0°C.

The pH values at which the absorption spectra were measured are indicated on the corresponding curves.

pH-dependent changes in the absorption spectra of Antho2a E292A at (A) 2.8 mM, (B) 28 mM, (C) 140 mM, and (D) 500 mM Cl− at 0°C.

The pH values are indicated on the corresponding curves in each graph.

Absorption spectra of (A) wild type Antho2a and (B) the Antho2a E292A mutant under different Cl− concentrations at pH 6.5 and 0°C.

Each colour indicates a different concentration of Cl−.