Applied Biochemistry and Microbiology, Vol. 35, ffo. /. 1999, pp. 29-17. Translated from Prikladnayti Biokhimiya i Mikrobialogiya, Vol. 35, No. 1,@  1999, pp. 34-42. Original Russian Text Copyright © /999 hy Mosin, Skluclnev, Shvatz.

Incorporation of [2,3,4,5,6-2H5]Phenylalanine,

[3,5-2H2]Tyrosine, and [2,4,5,6,7-2H5]Tryptophan

into the Bacteriorhodopsin Molecule of Halobacterium halobium

O. V. Mosin*, D. A. Skladnev**, and V. I. Shvets*

* Lotnonosov Moscow State Academy of Fine Chemical Technology, Moscow, 117571 Russia

** State Center of Genetics and Selection of Industrial Microorganisms (GNU GENETICA), Moscow, 113515 Russia

Received September 25, 1997

Abstract—Incorporation of [2,3A5,6-2H5]phenylalanine, [3,5-2H2]tyrosine, and [2,4,5,6,7-2H5]tryptophan into the bacteriorhodopsin molecule followed by semipreparative isolation of bacteriorhodopsin resulted in a yield of 8-10 mg per g bacterial biomass. This method is based on the growth of the strain of halophilic bacteria Halobacterium halobium on a synthetic medium containing 2H-labeled aromatic ammo acids and fractionation of solubilized (in 0.5% sodium dodecyl sulfate) protein by methanol, including purification of carotenoids. lip-ids, and high-molecular-weight and low-molecular-weight compounds, as well as gel-permeation chromatog-raphy on Sephadex G-200. Incorporation of 2H-labeled amino acids was analyzed by electron impact mass spectrometry after hydrolysis of the protein in 4 N Ba(OH)2 and separation in the form of methyl esters of /V-DNS derivatives of amino aids by re versed-phase high-performance liquid chromatography.

The retinal-containing protein (a chromophore, pro-tonated aldimine of retinal containing Lys-216 e-amino group) bacteriorhodopsin (BR), functioning as an ATP-dependent translocase in cell membranes of halophilic bacteria Halobacterium halobium was initially described by Oesterhelt [1]. In spite of the fact that the structure and functions of this protein were studied in detail, it is still a focus of interest. This protein is used in practice as a biological photochromic material because of its high photosensitivity and resolution abil­ities [2]. Moreover, BR is attractive as a model object for studies of the functional activity and structural properties of membrane proteins hi the composition of artificially designed energy-transforming membranes.

The introduction of isotopic labels into molecules of membrane proteins is appropriate for studies of these proteins. Isotopic labels allow using the method of high-sensitivity electron impact (El) mass spectrome­try for further analysis of isotopic incorporation [3, 4]. Thus, studies of BR labeled with the hydrogen isotope (deuterium) at residues of functionally important amino acids (phenylalanine, tyrosine, and tryptophan) involved in hydrophobic interaction of the protein polypeptide chain with the lipid bilayer of the cell membrane are important for practice [5, 6]. Raw 2H-labeled amino acids can be readily synthesized in pre­parative quantities by a reverse isotopic 1H-2H exchange in molecules of protonated amino acids, [2,3,4,5,6-2H5]phenylalanine (in 85% 2H2SC>4 at50°C), [3,5-2H2]tyrosine (in 6 N 2H2SO4 at slight boiling), and [2,4,5,6,7-2H5]tryptophan (in 75% [2H]trifluoroacetic acid at 25°C) [7]. However, in spite of the rapid devel­opment of chemical methods for obtaining 2H-labeled

aromatic amino acids, the Russian industry of individ­ual 2H-labeled membrane proteins has not received wide acceptance.

This work was designed to obtain sernipreparative quantities of 2H-labeled BR for reconstruction of artifi­cial membranes. Processes of incorporation of [2,3,4,5,6-2H5]phenylaIanine, [3,5-2H2]tyrosine, and [2,4,5,6,7-2H5]tryptophan into the molecule of bacteri­orhodopsin followed with further semipreparative iso­lation were performed. The deuteration level was deter­mined by means of El mass spectrometry performed after separation of the protein hydrolysate in the form of methyl esters of /V-DNS derivatives of amino aids by reverse-phase high-performance liquid chromatogra­phy (HPLC).

MATERIAL AND METHODS

Objects of studies. The carotenoid-contain ing strain of extreme halophilic bacteria Halobacterium halo-bium ET 1001 from the collection of cultures of micro­organisms (Moscow State University) was used. The strain was maintained on solid peptone medium (2% agar) containing 4.3 M NaCl.

Preparation of growth media. DL-amino acids (Reanal, Hungary), adenosine monophosphate (AMP) and uridine monophosphate (UMP) (Sigma, USA), were used. 5-[Dimethylamino]naphthalene-l-sulfonyl chloride (DNS chloride; Sigma, USA) and diaz-omethane obtained from JV-nitroso-Af-methylurea (Merck, Germany) were applied for the synthesis of amino acid derivatives. [2,3,4,5,6-2H5]Phenylalanine (90 at. % 2H), [3,5-2H2]tyrosine (96 at. % 2H), and

[2,4,5,6,7-2H5]tryptophan (98 at. % 2H) (methods for obtaining are described in [8, 9]) were supplied by A.B. Pshenichnikova (Candidate of Chemical Sci­ences, Lomonosov Moscow State Academy of Fine Chemical Technology).

2H-Labeled BR. 2H-Labeled BR was obtained on a synthetic medium, in which protonated ammo acids (phenylalanine, tyrosine, and tryptophan) were replaced by their deuterium-containing analogues ([2,3,4,5,6-2H5]phenylalanine, [3,5-2H2]tyrosine, and [2,4,5,6,7-2HJtryptophan). The medium contained 0.43 g/1 DL-alanine, 0.4 g/1 L-arginine,0.45 g/1 DL-aspartic acid, 0.05 g/1 L-cysteine, 1.3 g/1 L-glutamic acid, 0.06 g/1 L-glycine, 0.3 g/1 DL-histidine, 0.44 g/1 DL-isoleucine, 0.8 g/1 L-leucine, 0.85 g/1 L-lysine, 0.37 g/1 DL-methionine, 0.26 2/1 DL-phenylalanine, 0.05 g/1 L-proline, 0.61 g/1 DL-serine, 0.5 g/1 DL-thre-onine, 0.2 g/1 L-tyrosine, 0.5 g/1 DL-tryptophan, 1.0 g/1 DL-valine, nucleotides (0.1 g/1 AMP and 0.1 g/1 UMP), salts (250 g/I Nad, 20 g/1 MgSOa x 7H2O, 2 g/1 KC1, 0.5 g/1 NH4C1, 0.1 g/1 KNO3, 0.05 g/1 KH2PO4, 0.05 g/1 KoHPO4, 0.5 g/1 sodium citrate, 3 x 10 -4 g/1 MnSO4 x 2H2O, 0.065 g/1 CaCl2 - 6H2O, 4 x 10 -5 g/l ZnSO4 x 7H2O, 5 x 10 -5FeSO4 - 7H2O, and 5 x 10 -5 g/1 CuSO4 x 5H2O), 1 g/1 glycerin, and growth factors (1 x 10 -4 g/1 biotin, 1.5 x l0 -4 g/1 folic acid, and 2 x 10 -5 g/1 vita­min B!2).

Cultivation of bacteria. ............