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 - рефераты, скачать рефераты, бесплатно рефераты

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

b>Mass spectra. Mass spectra of methyl esters of N-DNS derivatives of amino acids were obtained by the method of electron impact on an MB-80 A instrument (Hitachi, Japan) at the energy of ionizing electrons of 70 eV, accelerating potential of 8 kV, and a temperature of the cathode source of 180-200°C. Scanning of the samples analyzed was performed at a resolution of 7500 conditional units and a 10% image definition.

RESULTS AND DISCUSSION

Incorporation of [2,3,4,5,6-2H5]phenylalanine, [3,5-2H2]tyrosine, and [2,4,5,6,7-2H5]tryptophan into the molecule of BR. The method of incorporation of 2H-labeled amino acids into the molecule of BR was selected because of the fact that this work was designed to reveal the possibility for obtaining 2H-labeled prepa-rations of the membrane protein (in semipreparative amounts) for the reconstruction of artificial membranes. [2,3,4,5,6-2H5]PhenyIalanine, [3,5-2H2]ryrosine, and [2,4,5,6,7-2H5;]tryptophan play important roles in hydrophobic interaction of the BR molecule with the lipid bilayer of the cell membrane. They are stable to the 'H-2H exchange in water medium under growth conditions. Moreover, high-sensitivity El mass spec-trometry can be used for the analysis of their incorpo-ration, which was performed microbio logically by growing the strain of halophilic bacteria Halobacte-rium halobium on a synthetic medium containing 2H-labeled aromatic amino acids. Thus, these compounds were selected as sources of deuterium. Under the opti-mum growth conditions (exponential growth on a syn-thetic medium with 4.3 M NaCl at 35-37°C and illumi-nation), the cells synthesized a purple pigment whose spectral characteristics were identical to those of native BR. Figure 1 shows the dynamics of (2) bacterial growth on the medium containing -H-labeled aromatic amino acids in relation to (1) growth under control con-

Fig. 1. The dynamics of Che growth of Che strain//, halobium under various experimental conditions: (/) protonated synthetic medium and (2) synthetic medium with [2,3,4,5,6-2H5]phenylalanine, [3,5-2H2Jtyrosine, and [2,4,5,6,7-2H5]tryptophan.

ditions. The growth of this strain on the medium con-taining 2H-Iabeled aromatic amino acids was only slightly inhibited. This is important for producing the raw 2H-labeled biomass for further isolation of BR.

The main stages of isolating 2H-labeled BR (Fig, 2) were the following: production of 1 g of 2H-labeled bio-mass; isolation of the fraction of PMs; removal of low-molecular-weight and high-molecular-weight admix-tures, cellular RNA, carotenoids, and lipids; fraction-ation of solubilized (in 0.05% SDS) protein by metha-nol; and purification on Sephadex G-200. Low-molec-ular-weight admixtures and the intracellular contents were eliminated by osmotic shock induced by distilled water (after removing 4,3 M NaCl) followed by destruction of cell membranes by ultrasound. The cel-lular homogenate was then treated with RNase I (two-three units of activity) to induce the maximum destruc-tion of cellular RNA. The PM fraction obtained con-tained the complex of the desired protein with Hpids and polysaccharides, as well as admixtures of fixed car-otenoids and foreign proteins. Therefore, it was neces-sary to use special methods of protein fracdonation, which would not damage the native structure of the pro-tein native structure or cause its dissociation. This made the isolation of pure individual BR performed by the use of special fine methods for removing carotenoids and lipids, purification, and column chromatography more difficult. Decarotenoidation was conducted by a repeated treatment of PMs with 50% ethanol at -5°C. Although it was a routine procedure, this stage was neces-sary (despite of considerable chromoprotein losses). The treatment was repeated no less than five times to obtain the absorption band of the PM suspension freed of caro-tenoids. Figure 3 shows (curves b, c) these bands at vari-ous stages of treatment in relation to (curve a) the band of

Growth of Halobacterium halobium on synthetic medium containing [2,3,4,5,6-2H5]phenyIalanine, [3,5-2H2]tyrosine and [2,4,5,6,7-2H5]tryptophan

Disintegration by ultrasound

Water-soluble products

of cellular content,

inorganic salts,

and other low-molecular-weight

compounds

Distilled H2O

RNase I,

125 mM NaCl, 20 mM MgCl,

4 mM Tris-HCl

Distilled H2O

Isolation of the biomass

Raw biomass
t

Osmotic shock

Culture liquid

4.3 M NaCl, and other

inorganic salts

and metabolites

50% ethanol

1.0.5%SDS-Na 2. Methanol

-5°C

PM fraction

Decarotenoidation

Delipidation + BR precipitation

-- Extract of carotenoids

_._ Residuals of cellular walls, lipids, and other high-molecular-weight compounds

Crystalline BR
t

Gel-permeation chromatography on Sephadex G-200

4NBa(OH)7 UO°C,24h

DNS chloride, 2 M
NaHCO3, and ethyl acetate

jV-Nitroso-N- methyl-

urea, 40% KOH

diethyl ester, and diazomethane

Purified BR ±

Mixture of free amino acids I

Modification into methyl esters

of /V-DNS derivatives of amino acids

Reverse-phase HPLC

BaSO4 after neutralization with 2 M 2 M H2SO4

Individual methyl esters of/V-DNS[2,3,4,5,6-2H5]phenylalanine

N-DNS-[3,5-2H2]tyrosine, and N-DNS [2,4,5,6,7-2H5]tryptophan

El mass spectrometry

Fig. 2. Experimentally designed method for isolating H-labeled BR.

native BR. In this case, an 80-85% efficiency of remov-ing carotenoids was reached. The formation of the reti-nal-protein complex induced a bathochromatic shift in the absorption band of PMs (Fig. 3). The major band recorded at the maximum absorption of 568 nm and induced by the light isomerization of chromophore at

bonds positioned at C13=C14 or multiples of this num-ber was determined by the presence of trans-retinal res-idue of retinal (BR568). The additional low-intensity band recorded at 412 nm characterized the presence of a minor admixture of the M412 spectral form (produced in light) containing the deprotonated aldirnine bond

between the residue of trans-retinal and the protein. The band recorded at 280 nm depended on the absorp-tion of aromatic amino acids of the polypeptide chain of this protein (the D2%0/D56% ratio was 1.5 : 1 for pure BR).

Fractionation and careful chromatographic purifica-tion of the protein were the next necessary stages. BR is a transmembrane protein with a molecular weight of 26.7 kDa that penetrates the lipid bilayer in the form of seven a-helixes. Therefore, the use of ammonium sul-fate and another traditional salt-eliminating agents is not appropriate. The protein must be transformed into the soluble form by solubilization in 0.5% SDS. The use of this ionic detergent was dictated by the necessity of the most complete solubilization of the protein achieved by combining delipidation and precipitation. In this case, BR solubilized in a low-concentration solution of SDS retained its helical cc-conformation [12]. Therefore, it was not necessary to use organic sol-vents such as acetone, methanol, and chloroform for removing lipids. Delipidation and precipitation of the protein were combined into the same stage. This noticeably simplified fracdonation. The advantage of this method was that the desired protein (in the com-plex with molecules of lipids and detergent) was in the supernatant. Another high-molecular-weight admix-tures were in the nonreacted precipitate, which was removed by centrifugation. Fractionation of solubilized (in 0.5% SDS) protein and its further isolation in the crystalline form were conducted using a gradual low-temperature (-5°C) precipitation by methanol (three stages). The second and the third stages were per-formed by decreasing the detergent concentration 2.5 and 5 times, respectively. The final stage of BR purifi-cation involved the separation of the protein from low-molecular-weight admixtures by gel-permeation chro-matography. The fractions containing BR were passed two times through a column with dextran Sephadex G-200 balanced with 0.09 M Tris-borate buffer (pH 8.35) con-taining 0.1% SDS and 2.5 mM EDTA. The method designed for fractionation of the protein made it possi-ble to obtain 8-10 mg of pure preparation of 2H-labeled BR from 1 g of bacterial biomass. The homogeneity of BR complied with the requirements on reconstruction of membranes and was confirmed by electrophoresis in 12.5% PAAG with 0.1% SDS, regeneration of apomembranes with trans-retinal, and reverse-phase HPLC of methyl esters of N-DNS derivatives of amino aids. Low yield of BR was no barrier to further studies of isotopic incorporation. However, it must be empha-sized that considerable amounts of the raw biomass must be produced in order to provide high yield of the protein.

Hydrolysis of BR. Conditions of hydrolysis of deu-terium-containing protein were determined by the necessity of preventing the isotopic ('H-2H) hydrogen-deuterium exchange in molecules of aromatic amino acids, as well as retaining tryptophan in the protein hydrolysate. Two alternative variants (acid and alkaline hydrolysis) were considered. Acid hydrolysis of the

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Fig. 3. Absorption bands (in 50% ethanol) at various stages of treatment: (a) native BR, (b) PMs after intermediate treat-ment, and (c) P.Ms purified of foreign admixtures. The band (/) corresponds to the spectral form of BR568. The band (2) corresponds to the admixture of the M^ spectral form. The band (J) characterizes the absorption of aromatic amino acids. The bands (4) and (5) correspond to foreign caro-tenoids. Native BR was used as control.

protein performed under standard conditions (6 N HC1 or 8 N H2SO4, 110°C, 24 h) is known to induce com-plete degradation of tryptophan and partial degradation of serine, threonine, and several other amino acids in the protein [13]. These amino acids do not play an important role in this study. The modification of this method involving the addition of phenol [14], thiogly-colic acid [15], and p-mercaptoethanol [16] into the reaction medium allowed retaining tryptophan (to 80-85%). 7-ToIuenesulfonic acid with 0.2% 3-(2-aminoet-hyl)-indole, as well as 3 M 2-mercaptoethanesulfonic acid [18], are the potent agents for retaining tryptophan (to 93% [17]). However, these methods are not suitable for working the problem, because they have a notice-able weakness. Processes of the isotopic exchange (of a high rate) of aromatic protons (deuterons) in mole-cules of tryptophan, tyrosine, and histidine [19], as well as the exchange of protons at C3 atom of aspartic acid and C4 atom of glutamic acid [20], proceed under con-ditions of acid hydrolysis. Thus, the data on incorpora-tion of deuterium into the protein can not be derived from the hydrolysis performed even in deuterium-con-taining reagents (2HC1,2H2SO4, and 2H2O).

Reactions of the isotopic hydrogen exchange are nearly undetected (except for the proton (deuteron) at C2 atom of histidine), and tryptophan is not degraded under conditions of alkaline hydrolysis (4 N Ba(OH)2 or NaOH, 110°C, 24 h). Thus, this method of hydroly: sis was used in our study. Simplification of the proce-dure for isolating the mixture of free amino acids (due

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500

600

Fig. 4. El mass spectrum of the mixture of methyl esters of /V-DNS derivatives of amino acids of the BR hydrolysate. Cultivation was performed on synthetic medium containing [2,3,4,5,6- Hslphenylalanine, [3,5- H2]tyrosine, and [2,4,5,6,7-2H5]tryptophan. Images of molecular ions of arnino acids correspond to their derivatives (here and on Fig. 5). Ordinate: relative intensity of the peak /)-

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