Biosynthetic incorporation of deuterium-labeled aromatic amino acids - [2,3,4,5,6-2H5]phenylalanine, [3,5-2H2]tyrosine and [2,4,5,6,7-2H5]tryptophan into the molecule of transmembrane protein bacteriorhodopsin from halobacterium Halobacterium halobium ET 1001
Authors: Oleg Mosin, Ignat Ignatov, Dmitry Skladnev, Vitaly Shvets
Number of views: 337
It was carried out the method for microbiological preparation and isolation of trans membrane photo-transforming protein bacteriorhodopsin (output 810 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 photoorganotrophic halobacterium Halobacterium halobium ET 1001 by cellular lysis by distilled water, processing of bacterial biomass by ultrasound, alcohol extraction of low and high-weight molecular impurities, cellular RNA, carotenoids and lipids, with 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 was proved by combination of preparative and analytical protein methods including elecrtophoresis in 12,5% PAAG with 0,1% SDS-Na, gel filtration chromatography on Sephadex G-200, reverse-phase HCLP, electron impact mass-spectrometry of methyl esters of N-5-(dimethylamino)naphthalene-1-sulfonyl chloride (dansyl) derivatives of amino acids.