A. V. Lipkin

Structure of the low molecular weight protein copurified with α-latrotoxin

Some samples of latrotoxin purified from the black widow spider venom contain two components: α-latrotoxin (Mr ∼ 130,000) and a low mol. wt protein with Mr about 8000. Clones carrying the cDNA sequence for the low mol. wt protein copurified with α-latrotoxin were isolated from spider venom glands. Nucleotide sequence analysis of the cloned cDNA revealed the primary structure of the polypeptide to be 18 amino acids signal peptide and 70 amino acids protein chain with mol. wt of 7947 and I of ∼ 4.0. The protein exhibits certain structural homology with erabutoxin-a from the sea snake.

Novel insecticidal toxins from the venom of the spider Segestria florentina

Three insecticidal polypeptide toxins (F5.5, F5.6, F5.7) with molecular masses 4973, 4993 and 5159Da were isolated from the venom of the central Asian spider Segestria florentina. These toxins caused the complete flaccid paralysis of Heliothis virescens (Lepidoptera: Noctuidae) larvae (LD(50) 4-10 microg/g), whereas they were inactive upon intravenous injections into mice. On the basis of N-terminal amino acid sequences a family of eight genes encoding highly homologues polypeptides (SFI1-SFI8) was revealed, some of which encode polypeptides actually demonstrated to be present in S. florentina venom. All deduced polypeptides consist of 46 amino acids residues. Comparison of primary structures of SFI1-SFI8 with other spider toxins suggests that this family might share structural and functional relationships with other small spider neurotoxins, several of which are known to be highly selective agonists/antagonists of different voltage-dependent Ca(2+) channels.

Structure and function of the potassium channel inhibitor from black scorpion venom

A novel inhibitor of K+ channels has been purified from the venom of the Central Asian scorpion Orthochirus scrobiculosus. For this polypeptide toxin (OsK- 1) with molecular mass 4205.7 Da complete amino acid sequence was determined by Edman degradation and C-terminal amino acid analysis, and was confirmed by cloning and sequencing of the toxin cDNA. OsK-1 consists of 38 amino acid residues and possesses high sequence homology with agiotoxin, kaliotoxin and some homology with other known K+-channel blockers from different scorpion venoms. The toxin was shown to block small-conductance Ca++-activated K+-channels in neuroblastomaxglioma NG 10815 hybrid cells (Kd =1.4 x M) which are insensitive to apamin and sensitive to charybdotoxin. The effect of OsK- 1 was reversible and concentration dependent.

Isolation, Purification, Crystallization, and Preliminary X-ray Diffraction Study of the Crystals of HU Protein from M. gallisepticum

HU proteins are involved in bacterial DNA and RNA repair. Since these proteins are absent in cells of higher organisms, inhibitors of HU proteins can be used as effective and safe antibiotics. The crystallization conditions for the M. gallisepticum HU protein were found and optimized by the vapor-diffusion method. The X-ray diffraction data set was collected to 2.91 Å resolution from the crystals grown by the vapor-diffusion method on a synchrotron source. The crystals of the HU protein belong to sp. gr. P41212 and have the following unit-cell parameters: a = b = 97.94 Å, c = 77.92 Å, α = β = γ = 90°.

From gene to structure: The protein factory of the NBICS Centre of Kurchatov Institute

The Protein Factory was established at the Centre for Nano, Bio, Info, Cognitive, and Social Sciences and Technologies (NBICS Centre) of the National Research Centre “Kurchatov Institute” in 2010. The Protein Factory, together with the Centre for Synchrotron Radiation and Nanotechnology, promote research on structural biology. This paper presents the technology platforms developed at the Protein Factory and the facilities available for researchers. The main projects currently being performed at the Protein Factory are briefly described.

Novel protein haponin regulates cellular response to oxidative stress

225 † A novel protein haponin (NP_115701.2) has been isolated and characterized earlier in the laboratory of hormonal regulation of proteins, Shemyakin– Ovchinnikov Institute of Bioorganic Chemistry, Rus sian Academy of Sciences [1]. Studies of haponin made it possible to express it in the baculovirus system, generate and purify polyclonal antibodies to haponin, which revealed the expression of the endogenous pro tein in cell lines of different origin, and to perform the search for protein partners of haponin [2].

Potentially implantable biocathode with the function of charge accumulation based on nanocomposite of polyaniline/carbon nanotubes

A potentially implantable biocathode with the function of charge accumulation based on a nanobiocomposite including multiwall carbon nanotubes, polyaniline, and bilirubin oxidase is developed. The regularities of the functioning of the obtained electrode are studied in air–saturated phosphate buffer solution, pH 7.4 (PB), and also in phosphate buffer solution containing redox–active blood components (BMB). The open circuit potential of the biocathode is 0.33 and 0.08 V vs. the saturated calomel electrode in PB and BMB, respectively; it is completely restored after at least three self-charge/discharge cycles with connection to resistors with different resistance. Bioelectrocatalytic current density of oxygen reduction is 0.50 and 0.42 mA cm–2 with the residual activity of 78 and 60% of the initial value after 12 h of continuous operation in PB at 25°C and in BMB at 37°C, respectively.

Identification of the ligand in the structure of the protein with unknown function STM4435 from Salmonella typhimurium

The unidentified ligand, which is present in the crystal of the protein with unknown function STM4435 from Salmonella typhimurium, was identified using a combination of high-resolution X-ray crystallography and accurate-mass time-of-flight mass spectrometry. The identified glycerol was present as a component of the solutions used for the isolation and crystallization of the protein and serves as the ligand mimicking the natural metabolite, presumably, 2-keto-myo-isonitol, which is indicative of the involvement of STM4435 in the myo-isonitol catabolism. The results of the present study show that this approach holds promise in complex studies aimed at determining, refining, or confirming the protein functions.

Truncated Variants of Serratia proteamaculans Oligopeptidase B Having Different Activities

Treatment of native psychrophilic oligopeptidase B from Serratia proteamaculans (PSP, 78 kDa) with chymotrypsin (soluble or immobilized on modified porous glass MPG-PA) in the presence of 50% glycerol leads to production of a truncated enzyme form (PSP-Chtr, ∼66 kDa), which retains activity toward the low molecular weight substrate of PSP, BAPNA, but in contrast to PSP, is active toward the protein substrate azocasein. It has been shown by MALDI-TOF massspectrometry that PSP-Chtr lacks the N-terminal region of the molecule that envelops the catalytic domain of PSP and supposedly prevents hydrolysis of high molecular weight substrates. It has also been established that the lacking fragment corresponds to the N-terminal highest rank element of the informational structure of PSP. This finding confirms the usefulness of the method of informational structure analysis for protein engineering of enzymes. A similar treatment of PSP with immobilized trypsin also led to production of a stable truncated enzyme form (PSP-Tr, ∼75 kDa) which lacked 22 C-terminal amino acid residues and completely lost enzymatic activity, presumably because of changes in the nearest environment of His652 of the catalytic triad.

A novel approach to studying the structural and functional properties of proteins with unknown functions

Three proteins from extremophilic bacteria—hypothetical monooxygenase from Deinococcus radiodurans, hypothetical nucleotidyl transferase from Thermotoga maritime, and hypothetical oxidoreductase from Exiguobacterium sibiricum—and the DJ-1 chaperone protein from Homo sapiens have been produced in Escherichia coli. The isolation and purification procedures developed for the recombinant proteins allowed us to achieve yields higher than 96%. Crystallization conditions enabling stable growth of crystals have been determined. X-ray experiments have been performed to test the quality of the crystals and the resolution achieved ranged from 1.2 to 1.8 Å.