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

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

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 growth rate test of <i>E</i>. <i>coli</i>.

(XLSX)</p

Underlying numerical data of characterization of AbHeR.

(XLSX)</p

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

Kinetics of DNA looping by Anabaena sensory rhodopsin transducer (ASRT) by using DNA cyclization assay

DNA cyclization assay together with single-molecule FRET was employed to monitor protein-mediated bending of a short dsDNA (~ 100 bp). This method provides a simple and easy way to monitor the structural change of DNA in real-time without necessitating prior knowledge of the molecular structures for the optimal dye-labeling. This assay was applied to study how Anabaena sensory rhodopsin transducer (ASRT) facilitates loop formation of DNA as a possible mechanism for gene regulation. The ASRT-induced DNA looping was maximized at 50 mM of Na+, while Mg2+ also played an essential role in the loop formation.BN/Chirlmin Joo La

Underlying numerical data of properties of IMVs containing rhodopsins.

(XLSX)</p

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

(XLSX)</p

Proton-pumping photoreceptor controls expression of ABC transporter by regulating transcription factor through light

Abstract Light is a significant factor for living organisms with photosystems, like microbial rhodopsin—a retinal protein that functions as an ion pump, channel, and sensory transduction. Gloeobacter violaceus PCC7421, has a proton-pumping rhodopsin gene, the Gloeobacter rhodopsin (GR). The helix-turn-helix family of transcriptional regulators has various motifs, and they regulate gene expression in the presence of various metal ions. Here, we report that active proton outward pumping rhodopsin interacted with the helix-turn-helix transcription regulator and regulated gene expression. This interaction is confirmed using ITC analysis (K D of 8 μM) and determined the charged residues required. During in vitro experiments using fluorescent and luciferase reporter systems, ATP-binding cassette (ABC) transporters and the self-regulation of G. violaceus transcriptional regulator (GvTcR) are regulated by light, and gene regulation is observed in G. violaceus using the real-time polymerase chain reaction. These results expand our understanding of the natural potential and limitations of microbial rhodopsin function