Extended Data Fig. 4: Dimer interface.
a, SEC–MALLS analysis of HeR, using Shimazu HPLC 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. The three chromatograms show the readings from the UV absorption, refractive index, and light-scattering detectors. The traces were normalized to the peak maxima. The cyan and red curves in the light-scattering chromatogram indicate the calculated molecular masses of the protein–detergent complex (Mc) and the protein (Mp), respectively. The UV extinction coefficient of the protein was calculated by PROTPARAM and assumed to be 1.624. The refractive index increments (dn/dc) of the protein and the detergent were assumed to be 0.185 and 0.144, respectively. This experiment was performed once. b, Molecular mass values determined by the SEC–MALLS experiment with the standard errors on fitting or calculated from the amino acid sequence. The experimental protein mass was determined to be about 61.75 kDa, corresponding to the theoretical mass of the His-tagged HeR dimer, 59.7 kDa. c, d, UV–visible CD (blue) and absorption (green) spectra of HeR in a lipid membrane (POPE/POPG) (c) and in DDM (d). These experiments were performed once. e, f, In-cell thrombin-digestion of extracellular loops. Schematic diagram of the thrombin-digestion sites in the thrombin-digestion mutants of the extracellular loops (e). Immunoblotting analysis of the mutants (f). Protein digestion patterns were detected by the S-tag antibody. These results revealed that the thrombin digested only ECL1, and not ECL2 and ECL3. This pattern indicated that the first loop blocked the access of thrombin to ECL2 and ECL3, consistent with the crystal structure and also with the inverted topology as compared to the type-1 rhodopsins. This experiment was performed twice, and the representative data are shown. g, The four-helix bundle is expanded and shown in detail, with the interacting residues within 4.2 Å depicted as stick models. h, Tomographic representation along the dimer interface viewed from the extracellular side, showing the high surface complementarity within the four-helix bundle interface. This interface consists of an extensive network of interactions involving 16 residues in TM4 and TM5. i, List of rhodopsin oligomerization states.
