Extended Data Fig. 5: Validation of dimer formation.

a, Gel filtration chromatogram of the purified HeR, using AKTA purifier and Superdex 200 increase 10/300 column equilibrated with buffer, containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 0.03% DDM. Black line indicates the absorbance at 280 nm. Dashed line indicates the peak fractions used in the SDS–PAGE analysis in b. This experiment was performed twice, and the representative data are shown. b, SDS–PAGE analysis of the gel filtration fractions in a. The left and right panels show the images of the Coomassie brilliant blue-stained and unstained gels, respectively. The band corresponding to the HeR with colour indicates that stable dimer formation without the dissociation of the retinal even in the SDS–PAGE. Both bands corresponding to the monomer and dimer are double, The HeR structure contains all the residues of HeR except for the N-terminal His₆ tag and the residues 1–3, denying the proteolysis of the purified HeR. We suppose that the double bands reflect the difference in denaturation by SDS. c, E. coli cells expressing the wild-type and deletion mutant of HeR. This experiment was performed twice, and the representative data are shown. d, Absorption spectra of the wild-type and deletion mutant of HeR solubilized with DDM from E. coli membrane, in the presence of 500 mM hydroxylamine. Black and coloured spectra were measured under dark and light conditions, respectively. These experiments were performed twice, and the representative data are shown. e, Dark-minus-light difference spectra of the wild-type and deletion mutant of HeR in d. The orange spectrum is multiplied by 5.0. f, Conservation of the surface residues of the HeR structure. The sequence conservation among 531 HeRs was calculated using the ConSurf server (http://consurf.tau.ac.il), and is coloured from cyan (low) to maroon (high). The residues constituting the dimer interface, especially those in ECL1, show low sequence conservation among the HeRs. g, Sequence difference in the residues constituting the dimer interface. The conserved and not-conserved residues between TaHeR and HeR 48C12 are coloured in dark turquoise and orange, respectively. 23 of the 30 residues constituting the interface are different between TaHeR and HeR 48C12. h, A typical HS-AFM image of HeR 48C12 dimers in a lipid membrane. The HS-AFM experiments were performed four times and similar images were obtained. Representative data are shown. i, An averaged image (50 frames) of HeR 48C12 dimers. Despite the sequence difference between TaHeR and HeR 48C12, the HS-AFM images revealed that HeR 48C12 also forms a dimer in the membrane.