Table S1. Homology indices of Bop I with other archaeal bacteriorhodopsins.

Figure S1. Visible CD spectra for claret membranes (full line) and solubilized Bop I retinal protein (dashed line). OD at 550 nm of claret membranes (in water) and solubilized Bop I (i.e. BR II, in OG buffer) were about 0.5.

Figure S2. SDS-PAGE of BR I and BR II bands. Samples (10 μg each) were separated on a 15% Tris-Tricine gel and the proteins visualized by Coomassie stain. M= molecular mass markers.

Figure S3. Expression of the Bop I and Bop II proteins. Total RNA from H. walsbyi cells was subjected to RT-PCR using two specific primer couples, for Bop I or Bop II respectively (lanes 3 and 6). Genomic DNA from H. walsbyi cells was amplified in parallel as positive controls (lanes 2 and 5). Lanes 1 and 4 are negative controls. M= molecular mass marker.

Figure S4. Titration of protonated Schiff base in solubilized Bop I retinal protein. Difference absorption spectra of BR II at (pH)i-pH 7.34. Spectra are shown for pH 7.65, 8.01, 8.43, 8.78, 9.51 and 10.44. Inset: Absorption spectra of BR II at pH 7.34 (solid line) and 10.44 (dotted line).

Figure S5. Neutral lipid analysis of claret membranes and solubilized Bop I retinal protein by TLC. Solvent B (hexane/diethyl ether/acetic acid, 70:30:1, by vol) was used. One hundred micrograms of both claret membrane and BR II total lipid extracts and ten micrograms of the lipid standard (ret= a mixture of retinal isomers; car= β-carotene; sq= squalene) were loaded onto the plate. Lipids were detected by spraying with 5% sulphuric acid, followed by charring at 120 °C.

Figure S6. Comparison of Bop I and Bop II aminoacid sequences with selected members of bacteriorhodopsin family. The annotated aminoacid sequences were aligned with ClustalW algorithm including (from top to bottom) (1) Bop I from H. walsbyi, (2) Xop I from Haloarcula marismortui, (3) aR1 from Halobacterium sp. aus-1, (4) aR3 from Halorubrum sodomense, (5) aR2 from Halobacterium sp. aus-2, (6) aR4 from Halobacterium sp. xz515, (7) BR from H. salinarum NRC-1, (8) Bop II from H. walsbyi. Seventy-six amino acids (*) are conserved and seventy-nine similar (: and .) in the aligned sequences, most of which are localized in helix C, containing D85 and D96 (in bold in the sequences), which are the proton acceptor and donor from and to the Schiff base, respectively. K218 (site of retinal binding) is also marked bold. The putative helices are shown with broken lines.

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