O.V. Mosin, V.I. Shvets, D.A. Skladnev, I.I. Ignatov

Bacteriorhodopsin (BR), named by analogy to protein of the visual apparatus of mammals  rhodopsin was allocated from a cellular membrane of extreme photoorganoheterotrophic halobacterium Halobacterium halobium in 1971 by W. Stoeckenius and D. Oesterhelt (USA). This phototransforming transmembraine protein has molecular mass 26,7 kDa, and by its structure represents chromoprotein, connected by a Shiff base (as in visual pigments of animals) with residue of Lys-216 which contains as a chromophore group responsible for its color equmolecular mixiture of 13-cis- and completely 13-tranc-retinol С20-carotenoid, defining purple-red colour of halophilic bacteria. Along with BR a cellular membrane of halobacteria contains in small amount other accompanying carotenoid pigments, the basic of which bacterioruberin, defines stability of halobacteria to sunlight. Polipeptide chain of BR consists from 248 amino acid residues, 67 % of which are hydrophobic, formed with participation of aromatic amino acids  phenylalanine, tyrosine and tryptophan. The BR molecule consists of seven segments in confirmation of α-spirals penetrating cellular membrane in a direction, perpendicular to its plane. Hydrophobic domains represent transmembraine segments, and hydrophilic domains protruding from cellular membrane and connect separate intramembraine α-spiral segments of protein molecule. We have carried out the microbial synthesis of BR with output 810 mg, labeled with deuterium on residues of [2,3,4,5,6-²H₅]phenylalanine, [3,5-²H₂]tyrosine, and [2,4,5,6,7-²H₅]tryptophan. The protein was allocated from membranes of halobacterium Halobacterium halobium by lysis of cells in the distilled water, processing by ultrasound at 22 kHz, alcohol extraction of low and high-weight molecular impurities, cellular RNA, carotenoids and lipids, the subsequent solubilization of final product with 0,5 % SDS-Na and fractionation by methanol. The homogenity of the synthesized product, and the selectivity of deuterium encorporation into the molecule of BR was proved by elecrtophoresis in 12,5 % PAAG with 0,1 % SDS-Na, gel filtration chromatography on Sephadex G-200, reverse-phase HPLC, electron impact mass-spectrometry of methyl esters of N-Dns-derivatives of amino acids.