Extended Data Fig. 1: Phylogenetic tree and biochemical characterization.
a, Phylogenetic tree of HeRs and representative type-1 rhodopsins. HeR 48C12, the first-discovered HeR shows 12.6% identity and 35.4% similarity with bacteriorhodopsin (BR), and 15.0% identity and 36.0% similarity with green-absorbing proteorhodopsin (GPR). b, Ion-transport assays of TaHeR, HeR 48C12, and GPR. This experiment was performed twice, and the representative data are shown. c, ATR–FTIR spectra on the exchange between Cl− and Br− in the solvent for TaHeR and the Cl− pump (NpHR). d, ATR–FTIR spectra on the exchange between Na+ and K+ in the solvent for TaHeR and the Na+ pump (KR2). This experiment was performed twice, and the representative data are shown. e, HPLC pattern of chromophores extracted from TaHeR. HPLC pattern of retinal extracted from TaHeR in the dark (blue) and under illumination at λ = 540 ± 10 nm (green). Most of the retinal (96%) bound to TaHeR adopts an all-trans configuration in the dark (n = 3). When the retinal was extracted after illumination, the proportion of the 13-cis form increased to 39% (n = 3). The representative data are shown, f, Deprotonation of the retinal Schiff base of TaHeR at an alkaline pH. Difference absorption spectra (left) and absorption change at λ = 542 nm (right, red solid circles) of TaHeR upon a pH change from 8.4 to 11.7 and higher values. The deprotonated form of the retinal Schiff base showed the absorption at λ = 392 nm. The acid dissociation constant (pKa) value in the right panel indicates mean ± s.d., which is fitted with the Henderson–Hasselbalch equation (grey dashed line). This experiment was performed once. g, Red-shift of the UV–visible absorption spectrum of TaHeR, and protonation of the counterion. The UV–visible absorption spectra (left) and the λmax (right, red solid circles) of TaHeR at pH 1.6–7.0. When the pH is lowered, a red-shift of the absorption is observed, and this is commonly reported for many type-1 rhodopsins and reflects the protonation of counterions. Thus, the red-shift of TaHeR originates from the protonation of Glu108, which is fitted with the Henderson–Hasselbalch equation (grey dashed line), and the pKa of the counterion (Glu108) is estimated to be 3.6. At pH values less than 2.1, a large blue-shift to 450 nm is observed, presumably due to the acid denaturation of the protein. The pKa value in the right panel indicates mean ± s.d. on fitting. This experiment was performed once.
