The venom of South American ant Paraponera clavata and its low-molecular-mass fraction were shown to possess insectotoxic and pore-forming activities. A number of glycophospholipid components were isolated from this ant venom by means of gel filtration and reversed-phase chromatography. Some of the compounds cause conductivity fluctuations in lipid bilayer membranes within the ranges 3–25 pS and 200–400 pS at concentrations of 10 −6 to 10 −7 M. N -Acetylglucosamine, a fatty acid, and phosphoric acid residues were found in their structures. A full structure, 3-myristoyl-2-acetamido-2-deoxy-α- D -glucopyranosyl phosphate, was elucidated for one of the compounds by the use of 1 H-, 13 C-, and 31 P NMR spectroscopy and mass spectrometry.

A green fluorescent protein from the coral Dendronephthya sp. (Dend FP) is characterized by an irreversible lightdependent conversion to a red-emitting form. The molecular basis of this phenomenon was studied in the present work. Upon UV-irradiation at 366 nm, the absorption maximum of the protein shifted from 494 nm (the green form) to 557 nm (the red form). Concurrently, in the fluorescence spectra the emission maximum shifted from 508 to 575 nm. The green form of native Dend FP was shown to be a dimer, and the oligomerization state of the protein did not change during its conversion to the red form. By contrast, UV-irradiation caused significant intramolecular changes. Unlike the green form, which migrates in SDS-polyacrylamide gels as a single band corresponding to a full-length 28-kD protein, the red form of Dend FP migrated as two fragments of 18- and 10-kD. To determine the chemical basis of these events, the denatured red form of Dend FP was subjected to proteolysis with trypsin. From the resulting hydrolyzate, a chromophore-containing peptide was isolated by HPLC. The structure of the chromophore from the Dend FP red form was established by methods of ESI, tandem mass spectrometry (ESI/MS/MS), and NMR-spectroscopy. The findings suggest that the light-dependent conversion of Dend FP is caused by generation of an additional double bond in the side chain of His65 and a resulting extension of the conjugated system of the green form chromophore. Thus, classified by the chromophore structure, Dend FP should be referred to the Kaede subfamily of GFP-like proteins.

С использованием соответствующего фрагмента кДНК и выращивания клеток Escherichia coli на синтетической среде с добавкой 5-фтортриптофана получен белок, отвечающий экстрацеллюлярному домену (остатки 1-209) α-субъединицы никотинового ацетилхолинового рецептора из электрического органа Torpedo californica. Наличие фрагмента (His)6, предшествующего последовательности 1-209, позволило провести очистку белка, выделенного из тел включения, на Ni-NTA-агарозе. Анализ гидролизата белка с помощью 19F-ЯМР показал, что включение остатков 5-фтортриптофана составило ~50%. Спектр белка, подвергнутого восстановлению в денатурирующих условиях и последующему реокислению в разбавленном растворе в присутствии 0.05% SDS, характеризуется достаточным разрешением, что позволило провести частичное отнесение сигналов 19F с использованием мутанта Trp60Phe. Способность полученных доменов специфически связывать α-нейротоксины змей показана с использованием радиоиодированного α-бунгаротоксина и трифторацетилированного α-кобратоксина.A protein corresponding to the extracellular 1-209 domain of the α-subunit of the nicotine acetylcholine receptor from the electric organ of Torpedo californica was prepared using the corresponding cDNA domain by culturing Escherichia coli cells on a synthetic medium supplemented with 5-fluoro-L-tryptophan. The presence of a (His)6 fragment preceding the 1-209 sequence allowed purification of the protein isolated from inclusion bodies by affinity chromatography on Ni-NTA Agarose. The incorporation of 5-fluorotryptophan residues was found by 19F NMR to be ~50%. The spectrum of the protein reduced under denaturing conditions and subsequently reoxidized in a dilute solution under denaturing conditions in the presence of 0.05% SDS was sufficiently resolved, which allowed partial assignment of 19F resonances using the Trp60Phe mutant protein. The ability of the prepared domains to specifically bind snake α-neurotoxins was demonstrated with the use of radioiodinated α-bungarotoxin and trifluoroacetylated α-cobratoxin.

Для выявления продуцентов сидерофоров термофильные и терморезистентные штаммы бацилл были скринированы на среде с хром-азуролом S. Обнаружено, что штамм Bacillus licheniformis VK21 резко увеличивает секрецию метаболита - хелатора ионов Fe в ответ на добавление солей марганца (II). Рост продуцента в минимальной среде с MnS04 в условиях лимита по железу сопровождался накоплением катехольного продукта, содержание которого достигало максимума в начале стационарной фазы роста культуры. Наличие в среде FeCl3 приводило к значительному снижению количества образующегося катехола. Сидерофор, названный SVK21, был выделен из культуральной жидкости и очищен с помощью обращенно-фазовой ВЭЖХ; его сидерофорная функция доказана тестом на восстановление роста клеток продуцента в среде с EDTA. УФ-спектр сидерофора имеет максимумы поглощения при 248 и 315 нм. По данным аминокислотного анализа и ЯМР-спектроме-трии, метаболит

Thermophilic and thermoresistant strains of bacilli were screened on a medium containing Chrome Azurol S for the producers of siderophores. It was found that the Bacillus licheniformis VK21 strain dramatically increases secretion of the metabolite, a chelator of Fe 3+ , in response to addition of manganese(II) salts. The growth of the producer on a minimal medium containing MnSO 4 under the conditions of iron deficiency is accompanied by the accumulation of a catecholic product, the content of which reaches maximum at the beginning of the stationary growth phase of culture. In the presence of FeCl 3 , the amount of the catecholic product in the medium considerably decreases. The siderophore, called S VK21 , 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 the amino acid analysis and NMR spectrometry, the metabolite S VK21 is 2,3-dihydroxybenzoyl-glycyl-threonine.

A protein corresponding to the extracellular 1–209 domain of the α-subunit of the nicotine acetylcholine receptor from the electric organ of Torpedo californica was prepared using the corresponding cDNA domain by culturing Escherichia coli cells on a synthetic medium supplemented with 5-fluoro- L -tryptophan. The presence of a (His) 6 fragment preceding the 1–209 sequence allowed purification of the protein isolated from inclusion bodies by affinity chromatography on Ni-NTA Agarose. The incorporation of 5-fluorotryptophan residues was found by 19 F NMR to be ∼50%. The spectrum of the protein reduced in the denaturing conditions and subsequently reoxidized in a dilute solution under denaturing conditions in the presence of 0.05% SDS was sufficiently resolved, which allowed partial assignment of 19 F resonances using the Trp60Phe mutant protein. The ability of the prepared domains to specifically bind snake α-neurotoxins was demonstrated with the use of radioiodinated α-bungarotoxin and trifluoroacetylated α-cobratoxin.

It was previously shown that the catalytic subunit of the plant toxin viscumin induces aggregation of small unilamellar liposomes and this process is inhibited by the mab_TA7 monoclonal antibody produced to the denatured catalytic subunit of viscumin (Agapov, I.I. et al. , FEBS Lett. , 1999, vol. 464, p. 63). The interaction of the synthetic F 101 –T 105 and A 96 –T 105 fragments of the viscumin catalytic subunit with the mab_TA7 monoclonal antibody was studied by 1 H NMR spectroscopy. Results of this study demonstrated that only the A 96 –T 105 fragment is capable of binding to mab_TA7. A nuclear Overhauser effect observed in the antigen–antibody complex and registered on the resonances of the free peptide transferred from the free state to the antibody-bound state was analyzed, the mab_TA7 antigen determinant (H 99 –T 105 ) was identified, and its conformation and orientation within the complex with the antibody were determined.

A protein corresponding to the extracellular 1-209 domain of the alpha-subunit of the nicotine acetylcholine receptor from the electric organ of Torpedo californica was prepared using the corresponding cDNA domain by culturing Escherichia coli cells on a synthetic medium supplemented with 5-fluoro-L-tryptophan. The presence of a (His)6 fragment preceding the 1-209 sequence allowed purification of the protein isolated from inclusion bodies by affinity chromatography on Ni-NTA Agarose. The incorporation of 5-fluorotryptophan residues was found by 19F NMR to be approximately 50%. The spectrum of the protein reduced under denaturing conditions and subsequently reoxidized in a dilute solution under denaturing conditions in the presence of 0.05% SDS was sufficiently resolved, which allowed partial assignment of 19F resonances using the Trp60Phe mutant protein. The ability of the prepared domains to specifically bind snake alpha-neurotoxins was demonstrated with the use of radioiodinated alpha-bungarotoxin and trifluoroacetylated alpha-cobratoxin.

The plant toxin viscumin (60 kD) consists of B- (“binding”) and A- (“active”) subunits joined by a disulfide bond. The B-subunit is a lectin interacting with galactose-containing glycolipids and glycoproteins of the cell surface. The A-subunit possesses N-glycosidase activity which modifies 28S ribosomal RNA. This results in irreversible inhibition of protein synthesis. After binding and receptor-mediated endocytosis viscumin-containing vesicles are transported to endoplasmic reticulum where the A- (catalytic) subunit is subsequently translocated to cytosol. It is possible that translocation of A-subunit requires its unfolding. For identification of epitopes which might appear during such unfolding, we developed hybridomas producing monoclonal antibodies against denatured viscumin A-chain. Resistance of hybridoma cells to cytotoxic action of viscumin suggests antibody–toxin interaction inside these cells. TA7 hybridoma cells against an epitope which appears only in denatured viscumin are insensitive to the toxin. This suggests that antibody–toxin interaction occurs before transmembrane translocation of the catalytic A-chain into the cytoplasm. Consequently, toxin resistance of TA7 hybridoma cells implies the appearance of a new epitope in viscumin during its intracellular transportation inside of vesicles. Sixty five octapeptides have been synthesized and epitopes have been identified for monoclonal TA7 antibody and immune mouse serum by means of ELISA. Based on the epitopic mapping the peptide A96-ETHLFTGT-T105 was chemically synthesized and binding of this peptide to the monoclonal antibody TA7 and conformation of antigenic determinant (L100-FTGT-T105) was investigated by means of 1H-NMR spectroscopy.

Effect of the monoclonal antibody (MAb) 5B6 produced to the solubilized preparation of bacteriorhodopsin on the protein photocycle was studied to examine conformational rearrangements on the surface of a functioning bacteriorhodopsin molecule. Using solid phase enzyme immunoassay, peptide phage display, and 1 H NMR spectroscopy, we demonstrated that the epitope recognized by MAb 5B6 is the Val69-Pro-Phe-Gly72 fragment of the protein, with the aromatic ring of Phe71 and the methyl groups of Val69 participating in the binding. MAb 5B6 exerted no significant effect on the photocycle of bacteriorhodopsin solubilized in Triton X-100 at pH 6.2 and 7.4 which suggested that, when functioning, bacteriorhodopsin retains the conformation and position of its Val69-Pro-Phe-Gly72 fragment.

Proton signals for nine synthetic peptide fragments of human interleukin-2 (region 59-78) were assigned for aqueous solutions both of pure peptides and their mixtures with LNKB-2 monoclonal antibody. The nonspecific magnetization transfer (NOE) between the antibody or its Fab-fragment and the peptides was studied upon large excess of free peptide over bound peptide. NOE spectra using modified pulse sequence, enabling to eliminate broad signals and achieve higher (peptide signal)/noise ratio were obtained. The saturation transfer experiments indicated that methyl groups of amino acid residues corresponding to Leu66,70,72, Val69 and Ala73 in interleukin-2 contact with the antibody binding site. Thus, the hydrophobic interactions are of major importance for the LNKB-2-IL-2 peptide complexes. The minimal IL-2 fragment which can still bind to LNKB-2 monoclonal antibody is -Leu70-Asn71-Leu72-.

An outline is given of some new NMR spectroscopic approachcs to the spatial structure of peptidcs in solution which have been recently suggested and tcsted in the Shemyakin Institute for Chemistry of Natural Products.The previously derivcd angular dcpendence of the peptidc vicinal 3 JNHCH coupling constant has been refined on the basis of the latest experimental data.It has hecn found that ion-dipole intcraction of thc type C=O • • • M + (wherc M + is an alkali metal ion) Ieads to a low ficld shift of thc 13 C signal of the carbonyl group.This effcct permits determination of the number and location of the Iigand groups which form thc intcrnal cavity of pcptidc and depsipeptide complcxones.The effect of 'shift reagents' on the NMR spectra shcds certain light on the spatial structure of peptides in solution, and, in particular, gives considerable information on the configuration of the amide bond and on the rotational states of the Ccr--C 11 bond.The INDOR and signals on combination frequency have been used for detection of 'hidden' signals (their multiplicity, chemical shift and splitting) and on thc assignment of the NH signals in the proton NMR spectra of peptides.V. F. BYSTROV, ET AL.We assume as before 1 that our dependence is expected in the form of a Karplus-like equation 3 J -

Die bei der Umsetzung der Selenophenketone (I) mit Butylnitrit (II) in Gegenwart von Natriumäthylat entstehenden Keto‐oxime (III) lassen sich mit Lithiumaluminiumhydrid zu den Aminoalkoholen (IV) reduzieren.

(2-Selenienyl)ethanolamines were synthesized by the reduction of the corresponding amino ketones and isonitroso ketones.

Substituted β-(2-selenienyl)ethylamines were synthesized by the reduction of the corresponding α,β-unsaturated nitro compounds. 2-(ω-Nitrovinyl)selenophene was alkoxylated and aminated.