Рефераты. 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

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

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 abil-ities [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 spectrome-try 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 pre-parative 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 devel-opment of chemical methods for obtaining 2H-labeled

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

This work was designed to obtain sernipreparative quantities of 2H-labeled BR for reconstruction of artifi-cial 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 bacteri-orhodopsin followed with further semipreparative iso-lation were performed. The deuteration level was deter-mined 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 chromatogra-phy (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 micro-organisms (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 Sci-ences, 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 vita-min B!2).

Cultivation of bacteria. The growth medium was autoclaved for 30 min at 0.5 atm (pH was brought to 6.5-6.7 using 0.5 N KOH). The inoculum was grown in 750-ml Erlenmeyer's flasks (the medium volume was 100 ml) on a 380-S orbital shaker (Biorad, Hungary) at 35-37°C under conditions of intensive aeration and illumination (three LDS-40 lamps of 1.5 Ix each). After 24 h, the inoculum (5-10%) was transferred to the syn-thetic medium and grown for five to six days (similarly to obtaining of the inoculum). All further manipula-tions for BR isolation were performed with the use of a dimming lamp equipped with an ORZh-1 orange light filter.

Isolation of the fraction of purple membranes (PM). The biomass (1 g) was washed with distilled water and precipitated on a T-24 centrifuge (Carl Zeiss, Germany) at 1500 g for 20 min. The precipitate was suspended in 100 ml of distilled water and kept at 4°C. After 24 h, the reaction mixture was centrifuged at 1500 g for 15 min. The precipitate was resuspended in 20 ml of distilled water, disintegrated by sonication (2 kHz, three times per 5 min) on a water bath containing ice (0°C), and centrifuged at 1500 g for 20 min. After washing with distilled water, the cellular homogenate was resus-pended in 10 ml of buffer containing 125 mM NaCl, 20 mM MgCl2, and 4 mM Tris-HCl (pH 8.0). RNase (5 u,g, two-three units of activity) was added. The mix-ture was incubated at 37°C. The same buffer (10 ml) was added 2 h later. The mixture obtained was kept at 4°C for 14-16 h. The water fraction was removed by centrifugation at 1500 g for 20 min. The precipitate of

PMs was treated (five times) with 7 ml of 50% ethanol at -5°C. The solvent was removed by centrifugation at 1200 g and cooling for 15 min. The protein concentra-tion was measured on a DU-6 spectrophotometer (Beckman, USA) calculating the D280/D56S ratio [10]. Regeneration of PMs was conducted as described in [11].

Isolation of BR. The fraction of PMs (1 mg/ml) was solubilized in 1 ml of 0.05% sodium dodecyl sulfate (SDS), kept at 37°C for 7-9 h, and centrifuged at 1200 g for 15 min. The precipitate was removed. Methanol (100 (ll) was added drop wise (three times) to the super-natant at 0°C. The mixture was kept at -5°C for 14-15 h and then centrifuged at 1200 g and cooling for 15 min. Fractionation was performed three times with decreas-ing the concentration of SDS to 0.2% and 0.1%. Crys-talline protein (8-10 mg) was washed with cold dis-tilled water and centrifuged at 1200 g for 15 min.

Purification of BR. This procedure was performed by gel-permeation chromatography on a calibrated col-umn (150 x 10 mm). Sephadex G-200 (Pharmacia, USA) served as the stationary phase (bed volume: 30-40 ml per g). The samples were taken manually. The column was balanced with the buffer solution contain-ing 0.1% SDS and 2.5 mM EDTA. The protein sample was dissolved in 100 p.1 of the buffer solution and eluted with 0.09 M Tris-borate buffer (pH 8.5, / = 0.075) and 0.5 M NaCl at a flow rate of 10 ml/cm2 per h. Combined protein fractions were subjected to lyo-philization.

Electrophoresis of the protein. The procedure was performed in 12.5% polyacrylamide gel (PAAG) con-taining 0.1% SDS. The samples were prepared for elec-trophoresis by standard procedures (LKB protocol, Sweden). Electrophoretic gel stained with Coomassie blue R-250 was scanned on a CDS-200 laser densitom-eter (Beckman, USA) for quantitative analysis of the protein level.

Hydrolysis of BR. The protein (4 mg) was placed into glass ampoules (10 x 50 mm in size), and 4 N Ba(OH)2 (5 ml) was added. The mixture was kept at 110°C for 24 h. The reaction mixture was suspended in 5 ml of hot distilled water and neutralized with 2 N H2SO4 to pH 7.0. The sediment of BaSO4 was removed by centrifugation at 200 g for 10 min, and the superna-tant was evaporated in a rotor evaporator at 40°C.

Synthesis of N-DNS derivatives of amino acids. DNS chloride (25.6 mg) in 2 ml of acetone was added gradually to 4 mg of dry hydrolysate of BR in 1 ml of 2 M NaHCO3 (pH 9-10) under conditions of constant mixing. The reaction mixture was kept at 40°C and mixing for 1 h, acidified with 2 N HCI to pH 3, and extracted (three times) with 5 ml of ethyl acetate. The combined extract was washed with distilled water to pH 7.0 and dried with anhydrous Na2SO4. The solvent was removed at 10 mmHg.

Methyl esters of N-DNS derivatives of amino acids. Wet N-nitroso-.N'-methylurea (3 g) was added to 20 ml of 40% KOH in 40 ml of diethyl ether and then mixed

on a water bath with ice for 15-20 min for obtaining diazomethane. After the completion of gas release, the ether layer was separated, washed with distilled water to pH 7.0, dried with anhydrous Na2SO4, and used for the treatment of /V-DNS derivatives of amino acids.

Separation of the mixture of methyl esters ofN-DNS derivatives of amino acids. This was performed by the method of reverse-phase high-performance liquid chro-matography on a Knauer liquid chromatograph (Ger-many) equipped with a Knauer pump, 2563 UV detec-tor, and C-R 3A integrator (Shimadzy, Japan). The col-umn of 250 x 10 mm in size was used. Separon C18 (Kova, Czech) served as the stationary reverse phase. The diameter of granules was 12 urn. The injection vol-ume was 10 mkl. The following systems of solvents were used: (A) acetonitrile and trifluoroacetic acid (at a vol-ume ratio of 100 : 0.1-0.5) and (B) acetonitrile. Gradi-ent elution processes were performed at a rate of 1.5 ml/min for 5 min (from 0% to 20% B), 30 min (from 20% to 100% B), 5 min (100% B), 2 min (from 100% to 0% B), and 10 min (0% B).

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