Extended Data Fig. 10: Characterization of HeRs in other domains of life and investigation of scrambling activity.

a, Phylogenetic tree of HeRs. The four HeRs investigated spectroscopically are shown in red. b, Alignment of the amino acid sequences of TaHeR, HeR 48C12, McHeR and EhvHeR. Secondary structure elements for α-helices and β-strands are indicated by cylinders and arrows, respectively. Conservation of the residues is indicated as follows: red panels for completely conserved; red letters for partially conserved; and black letters for not conserved. This alignment was done using ESPript3. c, d, The absorption spectra (c) and time evolution of the transient absorption changes (d) of HeR 48C12, TaHeR, McHeR, and EhvHeR, using laser flash photolysis. The absorption spectra were measured in 0.1% DDM for HeR 48C12, TaHeR and EhvHeR, and in 0.03% DDM for McHeR. McHeR and EhvHeR showed blue-shifted absorptions, as compared with those of HeR 48C12 and TaHeR. Transient absorption changes were measured with HeR 48C12 and TaHeR in the E. coli membrane fraction and McHeR and EhvHeR in 0.03% and 0.1% DDM, respectively. The pH values were 8.5, 8.0, 8.0 and 7.5 for HeR 48C12, TaHeR, McHeR and EhvHeR, respectively. These spectroscopy experiments were performed once. e, Schematic representation of the fluorescent NBD-labelled scramblase activity assay. f, Fluorescence traces of scrambling by protein-free (black), bovine opsin (red), bovine rhodopsin (pink), opsin of TaHeR (light green) and TaHeR (dark green), reconstituted in POPE:POPG (3:1) phospholipid liposomes. g, Quantification of the scrambling activities of bovine opsin, bovine rhodopsin, opsin of TaHeR and TaHeR. The data are the mean ± s.d. of three measurements. h, Model diagram representing the scramblase activities of HeR and bovine opsin (PDB code: 3CAP).