Photochemical and biochemical properties, as well as thermal and light stability of prepared IMV containing rhodopsins.

(A) The absorption spectra of AbHeR and empty vector IMV were measured. (B) H+-pumping in Gloeobacter rhodopsin (GR) WT IMV was measured using light-induced proton movement assay. The AbHeR WT IMVs were analyzed in the absence (gray color space) and presence of light (60 to 240 s). Black and red lines indicate reactions with and without CCCP, respectively. (C) AbHeR WT IMV was incubated in light (532 nm) at 55 μmol m-2s-1 and 37°C. This test was performed in an independent experimental group (n = 3). Data are the mean ± standard deviation. The underlying data of the graph can be found in S6 Data. (TIF)</p

Underlying numerical data of growth rate test of <i>E</i>. <i>coli</i>.

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Protein–protein docking to predict the 3D protein structure of AbHeR and AbGS.

AbHeR and AbGS are indicated as transparent cyan and red helices, respectively. The distances between the hydrogen bonds of interacting amino acids of AbHeR and AbGS were calculated by polar interaction tool in PyMOL and indicated using a yellow dotted line. The key active sites of AbGS are present in the 2 AbGS monomers of dodecamer, and the positions of the key active sites of the 2 different monomers in AbGS are indicated in yellow and white text. The positions of amino acids in AbHeR are indicated in blue text. (A, D, and E) Docking parts of key active sites in AbGS and AbHeR. (B and C) GS 3D structure (PDB: 6su3.1.A) in a position with bound Glu. (D and E) The positions of the docking prediction are indicated with white dotted circles. (B and D) Above and (C and E) top views of the positions are shown. (TIF)</p

Identification of AbGS binding to AbHeR.

(A) Relationship between microbial rhodopsin and heliorhodopsin. The unrooted maximum likelihood tree is shown. Blue oval, blue circle, red circle, and orange oval represent heliorhodopsins clade, reported heliorhodopsins, AbHeR, and heliorhodopsins containing GS gene, respectively. (B) Each eubacterium contains GS and heliorhodopsin arranged in operons in the indicated direction. Nucleotide gaps between HeR and GS are indicated. AbGS and AbHeR are indicated by orange and red arrows, respectively. Predicted GS and other heliorhodopsin are indicated by gray and blue arrows, respectively. Promoters in each operon are expressed by red bent arrows and predicted using phiSITE, Softberry, BDGP, and SAPPHIRE. (C) PPI between AbHeR and AbGS is determined by isothermal titration calorimetry analysis. The upper and lower panels represent raw data and enthalpy change per mol, respectively. AbGS was continuously added to AbHeR wild type. Nonlinear curves in the lower panels evaluated the best-fit curve. (D) Predicted model of PPI between AbHeR and AbGS. AbHeR dimers are embedded in the membrane, and the N- and C-termini of AbHeR are located in the CP side and EC side, respectively. AbGS dodecamer binds to AbHeR dimers and the potential of interactions between them is indicated by dotted black arrows. The underlying data of the graph can be found in S1 Data. ACR, anion channelrhodopsin; BR, bacteriorhodopsin; CCR, cation channelrhodopsin; CIR, Cl–-pumping rhodopsin; CP, cytoplasmic; EC, extracellular; GS, glutamine synthetase; HR, halorhodopsin; LR, Leptosphaeria rhodopsin; NaR, Na+-pumping rhodopsin; PPI, protein–protein interaction; PR, proteorhodopsin; SR I, sensory rhodopsin I; SR II, sensory rhodopsin II; VR, viral rhodopsin; XeR, xenorhodopsin; XR, xanthorhodopsin.</p

Topological, photochemical, biophysical, and hydrophobic characteristics of AbHeR.

(A) The membrane topology of AbHeR was predicted using the Philius server. The prediction reveals that AbHeR is a 7-transmembrane protein and its N-terminus is located on the cytoplasmic side. Transmembrane helix, extracellular (non-cytoplasmic) side, cytoplasmic side, and signal peptide are indicated in yellow, green, blue, and red colors, respectively. (B) Absorption spectra of AbHeR WT at different pH values. The absorption maxima at acidic, neutral, and alkaline pH were 570, 553, and 547 nm, respectively. (C and D) Differences of absorbance were calculated from the absorption spectra of AbHeR WT at different pH values. (E and F) The pKa values of counterion (E105) and retinal Schiff Baes were estimated using the Henderson–Hasselbalch equation. (G) AbHeR WT membrane vesicles were determined to exhibit no ion-pumping function through a light-induced proton movement assay. The mixture solution was composed of 20 mM each of LiCl, NaCl, KCl, CsCl, and Na2SO4 and was used to detect any ion pumping. The AbHeR WT membrane vesicle was measured in the absence (gray color space) and presence of light (60 to 240 s). Black and red lines indicate without and with CCCP, respectively. (H) Absorption maxima of purified AbHeR mutants at neutral pH are shown. The absorbance maxima were not significantly different compared to those of WT. (I) Hydrophobicity analyses were performed using ProtScale Tool (web.expasy.org/protscale), and amino acid scales were based on the Kyte and Doolittle method. The most frequently used scales are calculated based on hydrophobicity and hydrophilicity, and the secondary structure conformational parameter scales are calculated based on different chemical and physical properties of amino acids. Values of 0 on the y-axis are indicated by dashed gray. Heliorhodopsins, AbHeR, HeR-48C12, and Thermoplasmatales archaeon heliorhodopsin (TaHeR) were analyzed for similar hydrophobicity positions. Ion-pumping rhodopsins (BR, Halobacterium salinarum bacteriorhodopsin; GR, Gloeobacter violaceus rhodopsin; NaR, Krokinobacter eikastus Na+-pumping rhodopsin) were analyzed for different hydrophobicity positions among the ion-pumping rhodopsins. The heliorhodopsins contained hydrophobic residues in different positions. The underlying data of the graph can be found in S4 Data. (TIF)</p

Underlying numerical data of characterization of AbHeR.

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Underlying numerical data of properties of IMVs containing rhodopsins.

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Underlying numerical data of protein–protein interaction using isothermal titration calorimetry analysis.

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PPIs and binding affinities of AbHeR for AbGS.

(A) Multiple sequence alignments of AbHeR and predicted heliorhodopsin in the GS group were aligned using MUSCLE. Alignments of AbHeR with heliorhodopsin in the GS group. Negatively and positively charged residues near intracellular loops of heliorhodopsin are indicated by red and blue, respectively. (B–G) Dissociation constants of AbHeR for AbGS determined using ITC analysis. The upper and lower panels represent raw data and enthalpy change per mol, respectively. AbGS was continuously added to AbHeR mutants. Nonlinear curves of lower panels were evaluated by the best-fit curve. (H) Scheme of PPIs in each amino acid position of AbHeR for AbGS, as predicted by Protter. Positively and negatively charged residues on the cytoplasmic side are indicated as red and blue circles, respectively, with arrows. Dotted arrows indicate not expressed and solubilized proteins. Fold-changes for AbHeR mutants compared to WT indicate text with black arrows. (I) Predicted model of AbHeR function associated with AbGS. AbHeR dimers are embedded in the membrane, and the N- and C-termini of AbHeR are located on the CP and EC sides, respectively. AbGS dodecamer binds to AbHeR dimers and exhibits altered enzyme activity in the presence of light. The underlying data of the ITC analysis can be found in S1 Data. CP, cytoplasmic; EC, extracellular; GS, glutamine synthetase; ITC, isothermal titration calorimetry; MUSCLE, MUltiple Sequence Comparison by Log-Expectation; PPI, protein–protein interaction; WT, wild type.</p

Original images showing SDS–PAGE and western blot in S4D and S4E Fig.

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