[Catechol siderophore, produced by thermoresistent strain of Bacillus licheniformis VK21]. / Katekhol'nyi siderofor, produtsiruemyi termorezistentnym shtammom Bacillus licheniformis VK21.

Thermophilic and thermoresistant strains of bacilli were screened on a medium containing Chrome Azurol S for producers of siderophores. It was found that the Bacillus licheniformis VK21 strain dramatically increases secretion of the metabolite, a chelator of Fe3+, in response to addition of manganese(II) salts. The growth of the producer on a minimum medium containing MnSO4 under the conditions of iron deficiency is accompanied by the accumulation of a catechol product, the content of which reaches a maximum at the beginning of the stationary growth phase of culture. In the presence of FeCl3, the amount of the catechol product in the medium considerably decreases. The siderophore, called SVK21, was isolated from the cultural medium and purified by reversed phase HPLC, and its siderophore function was confirmed by the test for the restoration of growth of producer cells in a medium containing EDTA. The UV spectrum of the siderophore has absorption maxima at 248 and 315 nm. According to amino acid analysis and NMR spectrometry, the metabolite SVK21 is 2,3-dihydroxybenzoyl-glycyl-threonine. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.

[Synthesis and antibacterial activity of analogues of the N-terminal fragment of the sarcotoxin IA antimicrobial peptide]. / Sintez i antibakterial'naia aktivnost' analogov N-kontsevogo fragmenta antimikrobnogo peptida sarkotoksina IA.

Three 18-membered analogues of the N-terminal fragment of the sarcotoxin IA cationic antimicrobial peptide were synthesized by the solid phase method of peptide synthesis with the use of swellographic monitoring. The ability of these peptides to inhibit the growth of various bacteria in culture medium and their hemolytic activity in experiments on human erythrocytes were studied. The analogue completely corresponding to the N-terminal amino acid sequence of the natural sarcotoxin IA with the amide group on its C-terminus exhibited higher antibacterial activity. The presence of carboxyl group on the C-terminus or the substitution of Tyr for Trp2 resulted in a decrease in the antimicrobial activity of the peptide. Our results indicate that the amphiphilic N-terminal peptide corresponding to the 1-18 sequence of sarcotoxin IA involves the moieties responsible for the antimicrobial activity of the antibiotic.

[Secondary antimicrobial metabolites produced by thermophilic Bacillus spp. strains VK2 and VK21]. / Vtorichnye metabolity antimikrobnogo deistviia, produtsiruemye termofil'nymi shtammami Bacillus spp. VK2 i VK21.

A collection of thermophilic strains of the genus Bacillus was made. The strains were screened for antimicrobial activity. Strains VK2 and VK21 isolated from thermal springs of the Kamchatka Peninsula, and antagonistic to several gram-positive bacterial species were chosen for further investigation of antibiotics produced by them. Restriction analysis of DNA coding for 16S rRNA showed that both strains can be assigned to Bacillus licheniformis. It was shown that the lytic activity of strains VK2 and VK21 was not related to the synthesis of hydrolytic enzymes. The maximum level of antimicrobial activity in the growth medium was found to correspond to the beginning of the stationary growth phase. Addition of manganese sulfate induced sporulation and altered significantly the time course of antibiotic production in both strains. Active metabolites were extracted with n-butanol. They survived boiling for 30 min and were resistant to trypsin and chymotrypsin but were partly hydrolyzed by pronase. They were stable at a pH range of 2.0-9.0.

[Identification and cloning of peptide synthetase genes of thermostable bacilli using the polymerase chain reaction]. / Identifikatsiia i klonirovanie genov peptidil sintetaz termorezistentnykh batsill pri pomoshchi polimeraznoi tsepnoi reaktsii.

Fourteen thermophilic and thermostable strains of the genus Bacillus were studied. Total DNA was isolated from these strains and used as a template to identify and clone peptide synthetase genes by means of polymerase chain reaction. Amplified DNA fragments were cloned into a phasmid vector, and nucleotide sequences of cloned fragments were determined. Stringent thermophilic strains were shown to lack genetic systems, which are responsible for the synthesis of secondary metabolites and homologous to the known peptide synthetase genes. On the contrary, thermostable strains had peptide synthetases and produced antimicrobial secondary metabolites. Analysis of nucleotide sequences and deduced amino acid sequences of cloned PCR fragments from B. licheniformis strains VK2, VK21, and VK2101 showed that they are absolutely identical. The cloned DNA fragment was found to be a portion of the open reading frame, which we termed ORF1. Data from analysis of a partial nucleotide sequence of the peptide synthetase gene of strain VK21 indicated that a 9.5-kb region of chromosomal DNA contains sequences of two genes homologous to the B. subtilis peptide synthetase genes dhbB and dhbF. Strains VK2, VK21, and VK2101 were shown to synthesize siderophores. A method for screening bacteria with peptide synthetase genes has been developed.

[The dependence of stability of the green fluorescent protein-obelin hybrids on the nature of their constituent modules and the structure of the amino acid linker]. / Zavisimost' stabil'nosti gibridov zelenogo fluorestsentnogo belka s obelinom ot prirody sostavliaiushchikh ikh modulei i struktury aminokislotnogo linkera.

Recombinant plasmids containing genes for the green fluorescent protein (GFP) from Aequorea victoria and the photoprotein obelin from Obelia longissima linked in-frame by inserts differing in nucleotide sequences were constructed. The expression of the chimeric genes in Escherichia coli cells resulted in synthesis of the GFP-obelin hybrid proteins. These proteins were purified to homogeneity and subjected to limited trypsinolysis. It was shown that the resistance of GFP-obelin hybrid proteins to trypsin depends on the nature of their constituent modules and the amino acid sequences of linkers between the modules. The kinetics of accumulation of full-length hybrid proteins during the growth of bacterial cells does not depend on the structure of the peptide linkers. Most of the full-length product accumulates in cells in the form of inclusion bodies resistant to endogenous proteases. The soluble fraction of the protein undergoes considerable proteolysis regardless of the linker structure.

[Stabilized reaction mixture for in vitro mRNA translation]. / Stabilizirovannaia reaktsionnaia smes' dlia transliatsii mRNK in vitro.

Here we describe a method for obtaining a ready-to-use stabilized reaction mixture for in vitro translation of mRNA. We also demonstrate the stabilization of a complete translation mixture containing wheat germ extract, amino acids, ATP, GTP, creatine phosphate, creatine kinase, and the reaction buffer by lyophilization in the presence of various sugars. The greatest stabilizing effect is achieved by supplementing the mixture with 10% (mass/volume) trehalose, which is also a unique translation activator, enhancing the translation of various mRNAs. A lyophilized complete translation mixture containing trehalose can be stored at 4-8 degrees C for several months without losing its activity. The mixture can be easily reconstituted by adding an aqueous mRNA solution and retains the potential for reproducible functioning. This allows the employment of such a cell-free translation system for analytical screening of a broad spectrum of compounds inhibiting translation at various stages.

[Characteristics of N-terminal 60-kDa fragment of elongation factor 2]. / Svoistva N-kontsevogo 60-kDa fragmenta faktora élongatsii 2.

The N-terminal 60-kDa-fragment of elongation factor 2 from rat liver (EF-2) was obtained by the limited proteolysis of native EF-2 with elastase. This fragment consists of 506 N-terminal amino acid residues of EF-2. The conformational properties of both this fragment and EF-2 in solution were studied by circular dichroism and fluorescent spectroscopy. The contents of secondary structure components in the fragment and in the factor that were deduced from CD measurements agreed well with values predicted from their primary structures. Both proteins were resistant to denaturation with < or = 3 M urea and exhibited cooperative denaturation transitions. Temperature melting also proceeded cooperatively for the fragment and EF-2. Structural properties of the N-terminal 60-kDa-fragment are discussed in comparison with the biochemical characteristics and 3D structure of prokaryotic elongation factor EF-G.

[Biosynthesis and conformational state of 17-kDa and 27-kDa N-terminal fragments of elongation factor EF-2 in solution]. / Biosintez i konformationnoe sostoianie v rastvore 17-kDa- i 27-KDa- N-kontsevykh fragmentov faktora élongatsii EF-2.

N-Terminal fragments of the rat liver elongation factor EF-2 containing 162 (17 kDa) and 244 (27 kDa) amino acid residues of 857 (95 kDa) residues of the native protein were synthesized in E. coli cells and in a wheat germ cell-free translation system, and their conformations were studied. Both fragments were synthesized as inclusion bodies (nonspecific molecular aggregates). The conformations of the fragments in a solution were studied at neutral pH values by CD, fluorescence spectroscopy, scanning microcalorimetry, viscosimetry, gel-filtration, limited proteolysis, and interaction with monospecific anti-EF-2 antibodies and GroEL/ES molecular chaperone. Under nondenaturing conditions, both fragments existed in a solution as associates within a broad range of molecular masses, contained a considerable amount of elements of the intramolecular secondary structure, and represented globules without rigid tertiary structure (molten globules). A rigid tertiary structure was not formed even after the interaction of the fragments with the GroEL/ES molecular chaperone, thus indicating that the C-terminal fragment is essential for the formation of the rigid tertiary structure. Both fragments contained conformational antigenic determinants similar to those in the whole protein; i.e., despite the absence of the rigid tertiary structure, the fragments contained elements whose structure was similar to that of the corresponding regions in the whole protein.

[Structure-activity domain of elongation factor EF-2. Analysis of fragments of limited EF-2 hydrolysis, obtained using trypsin and elastase]. / Strukturno-funktsional'nye domeny faktora élongatsii EF-2. Analiz fragmentov ogranichennogo gidroliza EF-2, poluchennykh s pomoshch'iu tripsina i élastazy.

Limited hydrolysis of EF-2 with trypsin in mild conditions leads to cleavage at the N-terminal part of the protein, at the region of phosphorylation, at the Arg54 and Arg65 residues. The trypsinolysis product, fragment T1', containing Thr56 and Thr58, which are phosphorylated in EF-2, is also phosphorylated by EF-2-kinase at the same residues. In the phosphorylated EF-2, digestion by trypsin takes place only at Arg65, resulting in a reduction of the rate of hydrolysis in comparison with the native EF-2. Digestion of EF-2 with elastase results in the formation of two fragments E1 and E2 (60 and 40 kDa, respectively). Fragment E1 represents the N-terminal part of EF-2. It is resistant to the further action of elastase, is not cleaved by trypsin, and loses its capability for phosphorylation. Fragment E2, the C-end part of the molecule, is not resistant to the further action of elastase and retains its capability for ADP-ribosylation with the A fragment of diptheria toxin and NAD+. Electrophoretic analysis of EF-2 and its proteolytic fragments according to O'Farrell showed that the modification, resulting in the presence of two initial forms of EF-2, is located between the amino acid residues 66 and 506 of the polypeptide chain. In conclusion a possibility of studying the formation of partial functional activities within the framework of individual structure-functional domains using a set of N-terminal fragments of various length is discussed.

[Cloning and determination of the primary structure of cDNA, coding the central part of elongation factor 2 from the rat liver]. / Klonirovanie i opredelenie pervichnoi struktury kDNK, kodiruiushchei tsentral'nuiu chast' faktora élongatsii 2 iz pecheni krysy.

A number of cDNA clones coding for 417 amino acid residues of the central part of the rat liver elongation factor 2 (EF-2) have been isolated. The oligonucleotides complementary to EF-2 mRNA were used as primers for synthesis of the first strand of cDNA cloned. Structures of these oligonucleotides were determined in course of 3'----5' sequencing coding strand of EF-2 cDNA. This method of synthesis of specific cDNA enabled one to reduce essentially the number of recombinant clones to be screened for EF-2 cDNA. Comparative studies of deduced protein sequences of rat liver EF-2 and hamster EF-2 [1] revealed the only substitution of aspartate residue for glutamate residue (hamster EF-2). The homology between nucleotide sequences of rat and hamster EF-2 cDNA was 89%. Northern-blot analysis of rat liver poly(A)+ mRNA revealed the only species of mRNA 3000 nucleotides long. A strong stop-signal for reverse transcriptase in the 5'-region of rat liver EF-2 mRNA is discovered: probability of dissociation of the enzyme from mRNA is at least 97.5%.

[Primary structure of the elongation factor G from Escherichia coli. VI. Structure of peptides of cyanogen bromide cleavage of the G-factor molecule]. / Pervichnaia struktura faktora élongatsii G iz Escherichia coli. VI. Struktura peptidov bromtsianovogo rasshchepleniia molekuly G-faktora.

Peptides obtained as a result of cyanogen bromide cleavage of the G-factor have been studied. All 12 peptides embracing the whole structure of fragment T4 have been isolated. For their amino acid sequence determination, cyanogen bromide peptides have been further cleaved with trypsin, chymotrypsin, thermolysin, staphylococcal glutamic protease and BNPS-skatole. The complete primary structure of 9 from 12 cyanogen bromide peptides has been determined.

[Primary structure of the elongation factor G from Escherichia coli. V. Amino acid sequence of the C-terminal domain]. / Pervichnaia struktura faktora élongatsii G iz Escherichia coli. V. Aminokislotnaia posledovatel'nost' C-kontsevogo domena.

The amino acid sequence of the C-terminal domain of the elongation factor G (EF-G) has been studied. The polypeptide chain of the domain consists of 228 amino acid residues, and contains no tryptophan or cysteine residues. To determine its structure, the peptides obtained as a result of the fragment digestion by staphylococcal glutamic protease, cyanogen bromide cleavage, and tryptic hydrolysis of the fragment modified by maleic anhydride have been analyzed, as well as peptides obtained after hydrolyses of cyanogen bromide fragments with chymotrypsin, thermolysin and trypsin.

[Primary structure of the elongation factor G from Escherichia coli. VII. Study of peptides generated during hydrolysis of the T4 fragment by glutamic proteinase from Staphylococcus aureus]. / Pervichnaia struktura faktora élongatsii G iz Escherichia coli. VII. Issledovanie peptidov, poluchennykh pri gidrolize fragmenta T4 glutaminovoi proteinazoi Staphylococcus aureus.

For fragment T4, obtained on limited trypsinolysis of the G-factor, the amino acid sequence embracing 76% of its structure has been determined by analysis of peptides resulting from the fragment T4 cleavage with staphylococcal glutamic protease. These data permitted to assemble into one polypeptide chain 7 out of 12 earlier characterized cyanogen bromide peptides contained in the fragment T4.

[Primary structure of the elongation factor G from Escherichia coli. VIII. Structure of tryptic peptides comprising the T4 fragment of limited trypsinolysis of the G-factor]. / Pervichnaia struktura faktora élongatsii G iz Escherichia coli. VIII. Issledovanie struktury peptidov tripticheskogo gidroliza, vkhodiashchikh v sostav fragmenta T4 ogranichennogo tripsinolizata G-faktora.

Products of tryptic hydrolysis of the maleic anhydride modified fragment Th3 from limited thermolytic hydrolysis of the G-factor have been studied. Some short peptides which result from the trypsin action on the native G-factor molecule and belong to the fragment T4 obtained on limited trypsinolysis of the G-factor have been separated and their structure has been studied. As a result amino acid sequence has been determined by tryptic peptides containing 322 amino acid residues of the fragment T4 which makes up about 94% of its polypeptide chain.

[Primary structure of the elongation factor G from Escherichia coli. IX. Structure of peptides generated by cyanogen bromide cleavage of the G-factor isolated on thiol-activated sepharose and of the products of the G-factor cleavage at Asp-Pro bonds. Complete primary structure]. / Pervichnaia struktura faktora élongatsii G iz Escherichia coli. IX. Issledovanie struktury peptidov bromtsianovogo rasshchepleniia G faktora, vydelennykh na tiol-aktivirovannoi sefaroze, i produktov rasshepleniia G-faktora po sviaziam Asp-Pro. Polnaia pervichnaia struktura.

The amino acid sequence of cysteine- and cystine-containing peptides resulting from cleavage of the G-factor by cyanogen bromide has been determined. For structure analysis cyanogen bromide peptides were further degradated using trypsin, chymotrypsin, thermolysin, staphylococcal glutamic protease, or limited acid hydrolysis. The products of the G-factor cleavage at Asp-Pro bonds were also studied. The obtained data together with those published earlier permitted to establish the complete primary structure of the elongation factor G. The polypeptide chain consists of 701 amino acid residues and has molecular mass of 77321,46.

[Properties and role of tryptophan residues in the polypeptide chain of elongation factor G from E. coli]. / Svoistva i rol' ostatkov triptofana polipeptidnoi tsepi faktora élongatsii G E. coli.

Using chemical modification and spectrofluorimetry, it was shown that two tryptophane residues of the elongation factor G (EF-G) in positions 51 and 71 from the N-terminus are located on the surface of the EF-G molecule. The tryptophane residue in position 71 is effectively shielded against modification at binding of EF-G guanyl nucleotides. Modification of these tryptophane residues does not result in a loss of the nucleotide-binding activity but completely inhibits EF-G binding to the ribosome. Polarized fluorescence study showed that the relaxation properties of these exposed tryptophane residues essentially depend both on the presence of the C-terminal domain and on binding of nucleotides in the nucleotide-binding site located in the N-terminal part of EF-G. It was assumed that the C-terminal domain, the nucleotide-binding site and the site responsible for the EF-G binding to the ribosome are brought together in the three-dimensional structure of the elongation factor G.

[Ribosomal proteins of Escherichia coli. Some features of the primary and three-dimensional structure]. / Ribosomnye belki Escherichia Coli. Osobennosti pervichoi i prostranstvennoi struktury.

Some properties of E. coli ribisomal proteins, e. g. molecular weights, isoelectric points, amino acid composition, are reviewed. The three-dimensional structure of the proteins in solution has been studied and the difficulties and discrepancies arising from interpretation of the results obtained have been revealed. The peculiarities of the amino acid sequences typical of the majority of the proteins or the amino acid sequences presenting unique features are interpreted in terms of the primary structure of some ribosomal proteins. The primary structure of the proteins determined with the use of interesting methodological procedures is discussed.