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The Oct-1 (POU2F1) transcription factor is one of the most important regulatory proteins in humans and other mammals. An increase in Oct-1 aids the resistance to oxidative and cytotoxic stresses and radiation exposure. A high level of Oct-1 is found in many human tumors and correlates with low survival. Oct-1 interacts with its binding sites as a monomer, a homodimer, or a multimer. The nucleotide sequence of the Oct-1 binding site determines the character of interaction and the conformation of Oct-1 on target DNA, thus influencing the binding of Oct-1 co-repressors and co-activators. Nucleotide substitutions were introduced in all positions of the PORE and MORE sequences and tested for effect on the Oct-1 capability of forming monomeric and dimeric DNA-protein complexes. The position and nature of nucleotide substitutions were found to affect the type of Oct-1 binding to DNA. Several substitutions suppressed the formation of dimers, while others stimulated the process. Certain nucleotide substitutions completely prevented the binding of both monomers and dimers. The Oct-1 concentration in the cell is another factor that affects the character of DNA-protein interactions. Based on the results, the nature and affinity of interaction with Oct-1 is possible to predict from the nucleotide sequence for PORE and MORE sites of the human genome.

Seasonal and highly infectious strains of the influenza A and influenza B viruses cause millions of cases of severe complications in elderly people, children, and patients with immune diseases each year. Immunoglobulin A (IgA), which is an active component of humoral immunity, can prevent the spread of the virus in the upper respiratory tract. The preparation and study of the properties of recombinant virus-specific IgA could be an important approach to finding new means of preventing and treating influenza. Based on CHO DG44 cells, we developed stable monoclonal cell lines that produce monomeric and dimeric antibodies FI6-IgA1 and FI6-IgA2m1 to hemagglutinin (HA) of the influenza A virus. When studying the productivity, growth, and stability of the obtained clones, we found that the dimeric form of antibodies of IgA1 isotype is superior to other forms. The dimeric form of IgA antibodies plays a key role in mucosal immunity. Recognizing the prospects of using dimeric IgA as prophylactic and therapeutic mucosal drugs for viral infections, we studied their virus-neutralizing and antiviral activities on MDCK cell culture and compared them with the antibodies of the IgG1 isotype. This study presents the data on antiviral and virus-neutralizing activities of the FI6-IgA1 dimers to seasonal and highly infectious strains of influenza A virus.

A study of the induction of broadly neutralizing antibodies (bNAbs) in HIV-infected patients and vaccinated subjects revealed the main criteria for the formation of bNAbs (the duration of exposure to a viral antigen, the effect of the diversity of HIV variants, and the removal of barriers associated with the Env-dependent defense mechanisms of HIV-1). Modified trimers of the HIV-1 envelope protein (Env) exposed on virus-like particles (VLP) have unique properties: they (i) modulate the exposure of binding sites (bs) of the CD4 receptor and co-receptor; (ii) create steric restrictions for contact with bNAbs; (iii) increase the Env presentation density, thus enhancing the immune response; (iv) form stable trimers that do not induce off-target immune responses; and (v) allow additional modifications to their structure and construction of a platform with immunostimulating molecules. Immunization using a heterologous subtype-cross prime-boost regime with modified trimeric Env is capable of inducing somatic hypermutation levels necessary for the formation of bNAbs.

The biosynthesis of L-lactate oxidase in the Yarrowia lipolytica yeast during submerged cultivation in laboratory bioreactors ANKUM-2M has been studied. It has been shown under optimal conditions of yeast cultivation with L-lactate that 24.5 U/L enzyme accumulated in the medium and the yield was 2.0 U/(L h). An increase in the biosynthesis of L-lactate oxidase to 75 U/L and the yield to 3.2 U/(L h) was achieved in the medium with L-lactate (1%) and glucose (2%). The enzyme was purified 251 times to homogeneity by hydrophobic and ion exchange chromatography state with a yield of 45% and a specific activity of 55.3 U/mg. Techniques of gel filtration and denaturing electrophoresis showed that L-lactate oxidase from Y. lipolytica is a tetramer with a molecular mass of 200–230 kDa. The enzyme showed a strict specificity to L-lactate and did not oxidize fumarate, pyruvate, succinate, ascorbate, dihydroxyacetone, glycolate, D-lactate, D, L-2-hydroxybutyrate and D, L-alanine or D-serine.

This review summarizes modern data on the structure and functions ofheteromersformed by D1 and D2 dopamine receptors focusing on their role in the mechanisms of drug dependence. This article discusses potential functional significance of heterodimeric D1-D2 dopamine receptorsdue to their localization in the brain as well as unique pharmacological propertiesversus constituent monomers. It is shown that heteromerization results in dramatic changes in activated signaling pathways compare to the corresponding monomers. These studies update our current knowledge of ligand-receptor interactions and provide better understanding of dopamine receptors pharmacology. Furthermore elucidation of significance of heterodimeric D1-D2 dopamine receptors as drug targets is important for the development of new effective drug addiction treatment.

An ideal protective HIV-1 vaccine can elicit broadly neutralizing antibodies, capable of preventing HIV transmission. The strategies of designing vaccines include generation of soluble recombinant proteins which mimic the native Env complex and are able to enhance the immunogenicity of gp120. Recent data indicate that the cytoplasmic tail (CT) of the Env protein has multiple functions, which can affect the early steps of infection, as well as viral assembly and antigenic properties. Modifications in the CT can be used to induce conformational changes in functional regions of gp120 and to stabilize the trimeric structure, avoiding immune misdirection and induction of non-neutralizing antibody responses. Env-trimers with modified CTs in virus-like particles (VLPs) are able to induce antibodies with broad spectrum neutralizing activity and high avidity and have the potential for developing an effective vaccine against HIV.

The structure of three forms of a dimeric enzyme, human inosine triphosphate pyrophosphatase, is considered to identify the enzyme conformation changes causing the inactivation effect of a P32T mutation. Analysis of a nanosecond molecular dynamics is performed; the mean square deviations of the atoms between the wild-type and mutant homodimers, and also the heterodimer are calculated. A 3 ns modeling shows a greater displacement of atoms in mutant protomers. During molecular dynamics simulation, the strongest changes are observed in the loop between α2 and β2 (amino acid residues 28-33, an area of the P32T mutation), the loop between β5 and β6, and the C-terminal amino acid residues. The loop between (α2 and β2 has two conformations characterized by different positions of the Phe31 aromatic group. The distance between Cys33 (Cα) and Phe31 (C(z)) for wild-type and mutant protomers was -9 and 5.5 Å, respectively. These conformations were kept constant.

The computer model for a dimer of inulinase from Aspergillus ficuum is designed. The permolecular organization of inulinase from Aspergillus niger is experimentally investigated. The question about the role of various inulinase forms in manifestation of their functional activity is discussed. It is shown, that in the process of inulinase dimerization when contacts between monomeric forms of the enzyme are formed, a key role belongs to the nonpolar amino acid residues.

The immune-enzyme analysis was applied using anti-CD38 monoclonal antibodies to detect serum content of total (sCD38) and oligomeric fractions of soluble molecules CD38 (ol.sCD38) in patients with cervix cancer and malignant neoplasms of uterine body. The three- and four-fold increasing of serum level of sCD38 is demonstrated both under cervix cancer and malignant neoplasms of uterine body. The level of ol.sCD38 decreased in patients with cervix cancer and increased in case of patients with malignant neoplasms of uterine body. The serum content of ol.sCD38 was increased under adenocarcinoma of uterus and glandular epidermoid cancer. The content of sCD38 increased under adenocarcinoma of uterus but decreased under sarcoma of uterus. The highest level of soluble molecules of CD38 was established in patients with highly differentiated tumors and under invasion into endometrium but not into miometrium. On the whole, alterations in content of soluble molecules CD38 are associated with characteristics of tumor process that indicates at their monitoring significance under malignant neoplasms of uterus.

Interaction of intranuclear β-amyloid with DNA is considered to be a plausible mechanism of Alzheimer's disease pathogenesis. The interaction of single- and double-stranded DNA with synthetic peptides was analyzed using surface plasmon resonance. The peptides represent the metal-binding domain of β-amyloid (amino acids 1-16) and its variants with chemical modifications and point substitutions of amino acid residues which are associated with enhanced neurotoxicity of β-amyloid in cell tests. It has been shown that the presence of zinc ions is necessary for the interaction of the peptides with DNA in solution. H6R substitution has remarkably reduced the ability of domain 1-16 to bind DNA. This is in accordance with the supposition that the coordination of a zinc ion by amino acid residues His6, Glu11, His13, and His14 of the β-amyloid metal-binding domain results in the occurrence of an anion-binding site responsible for the interaction of the domain with DNA. Zinc-induced dimerization and oligomerization of domain 1-16 associated with phosphorylation of Ser8 and the presence of unblocked amino- and carboxy-terminal groups have resulted in a decrease of peptide concentrations required for detection of the peptide-DNA interaction. The presence of multiple anion-binding sites on the dimers and oligomers is responsible for the enhancement of the peptide-DNA interaction. A substitution of the negatively charged residue Asp7 for the neutral residue Asn in close proximity to the anion-binding site of the domain 1-16 of Aβ facilitates the electrostatic interaction between this site and phosphates of a polynucleotide chain, which enhances zinc-induced binding to DNA.

Size exclusion chromatography was used to assess the relative size of intact and diphenylamine-treated (DPA, with suppressed carotenoid synthesis) peripheral light-harvesting complexes (LH2 complexes) of the sulfurbacterium Allochromatium minutissimum. Both LH2 complexes were nonamers and had the same elution volume V(e), coinciding with that for the LH2 complex of Rhodoblastus acidophilus (strain 10050). Their molecular mass was 150 kDa. Bot pheophytinization of bacteriochlorophyll (BChl) at low pH and treatment with the detergent LDAO, affecting the hydrophobic interactions between the neighboring protomers, result in the fragmentation of the ring of the isolated LH2 complexes and formation of 55-kDa fragments with molecular masses corresponding to one-third of the initial value. Fragmentation caused by both pheophytinization and detergent treatment was much more rapid in DPA-treated LH2 complexes than in the intact ones. The 55-kDa fragments formed at low pH values contained monomeric bacteriopheophytin, while the fragments of a similar molecular mass formed at pH 8.0 in the presence of the detergent contained monomeric BChl. The observed fragmentation was hypothesized to reflect the inherent C3 symmetry of the LH2 complexes, with the preliminarily assembled trimers used as building blocks.

In cyanobacteria, phycobilisomes (PBS) act as antennae of the photosynthetic pigment apparatus. They contain brightly colored phycobiliproteins (PBP) and form giant supramolecular complexes (up to 3000-7000 kDa) containing 200 to 500 phycobilin chromophores covalently bound to the proteins. Over ten various PBP are known, which fall into three groups: phycoerythrins, phycocyanins, and allophycocyanins. Hollow disks of PBP trimers and hexamers are arranged into cylinders by colorless linker proteins; the cylinders are then assembled into PBS. Typical semidiscoid PBS consist of a central nucleus formed by three allophycocyanin cylinders and of six lateral cylinders consisting of other PBP and attached as fans to the nucleus. The PBS number, size, and pigment composition in cyanobacteria depend on illumination and other ambient factors. While PBS have certain advantages compared to other antennae, these pigment-protein complexes require more energy than the chlorophyll a/b- and chlorophyll a/c-proteins of oxygenic photosynthetic organisms.

The change in temperature is one of the factors affecting the activity of enzymes. In this work thermal denaturation and aggregation of cytochrome P450 BM3 were studied by atomic force microscopy. To determine specific temperature transitions the fluorescence analysis was used. In the low melting temperature range, 10-33 degrees C, a decrease in the fluorescence intensity of aromatic residues was observed with an increase in the fluorescence intensity of flavin groups. Protein melting in this range indicated three narrow S-shaped cooperative transitions at temperatures 16, 22 and 29 degrees C. Atomic force microscopy analysis in this temperature range showed that the shape of BM3 molecules remained globular in the form of compact objects (heights h < 7 nm, lateral dimensions d < 50 nm), but protein oligomeric state changed. The first two transitions were accompanied by a decrease in the degree of oligomerization and the third one was accompanied by its increase.

It is considered that sister chromatids are held together immediately after replication by special protein complex--cohesin that consists of Smc1--Smc3 core dimer and two additional subunits, Scc1 and Scc3. This process is called cohesion. We have characterized binding of cohesin complex to early- and late-replicated chromatin at different stages of the cell cycle in human cells HeLa and HT1080 using superresolution microscopy (based on Structural ilumination microscopy--SIM) and immunoelectron microscopy. It has been shown that cohesins do not play important role in cohesion of heterochromatic domains, but they provide cohesion and organization of subdomains in euchromatic regions.

In view of the amyloid hypothesis of Alzheimer's disease (AD), the key molecular event is the structural transition of Β-amyloid from the physiologically normal monomer state to soluble neurotoxic oligomers accumulating in the form of insoluble extracellular aggregates (amyloid plaques) in brain tissues. Zinc ions are known to play a crucial role in the formation of these pathological aggregates. The authors and collaborators have identified that the certain chemical modification and point amino acid substitutions in the metal-binding domain play a critical role in the formation of neurotoxic zinc-dependent oligomers and induce the development of cerebral amyloidosis and other pathological processes characteristic of AD. The results allow to use these forms of Β-amyloid as potential biomarkers of early diagnosis of AD. Zinc-dependent dimerization and oligomerization of Β-amyloid can be used as drug target for treatment of AD.

Survivin is an oncofetal protein involved both in inhibiting of apoptosis and in cell cycle regulation. The functions of survivin are defined by its structural state. Due to nature polymorphism, survivin cancontain either E or K amino acid in 129 residue, and K129 is commonly acetylated. Only the protein having acetylated K129 tends to form dimeric structure. Thus, antibodies detecting the amino acid substitution can be a useful tool for structural and functional research of the protein. To obtain the antibodies specific to amino acid substitution E129/K129 peptide fragments overlapping 129 amino acid residue were synthesized, rabbits were immunized with the peptides and affinity purification of the antibodies on sepharose conjugated with the peptides was carried out. The data of ELISA and western blot showed that antibodies obtained were able to detect amino acid substitution E129/K129 in the recombinant and endogenous survivin.

The crystal structure of the dimeric green fluorescent protein EGFP-K162Q with C-terminal deletion MDELYK (EGFPv) has been determined in space group P6 at resolution 1.34 A. The obtained structure has been compared with that of the monomeric form of EGFP (green biomarker with enhanced photophysical properties) determined in other crystal space group P2(1)2(1)2(1) at resolution 1.50 and 1.35 A [1, 2]. Two subunits in the EGFPv structure are packed at 75 degrees with the contact surface approximately 800 A2. The dimeric structure is stabilized by six hydrogen bonds and the central hydrophobic core built of six residues. The RMSD value for Calpha atoms of 3-230 residues in the superimposed P61 and P2(1)2(1)2(1) structures is 0.55 A. The distinguishing feature of EGFPv- P6(1) structure, compared with that of EGFP-P2(1)2(1)2(1), is the noticeable difference in orientation of the Glu222 side chain and also new conformation of the loop fragment 155-159 with deviations among the Calpha atoms of superimposed structures reaching for Lys156 - 4.6 A and Lys158 - 5.5 A

Glycine receptor is the anion-selective channel, providing fast synaptic transmission in the central nervous system of vertebrates. Together with the nicotinic acetylcholine, GABA and serotonin (5-HT3R) receptors, it belongs to the superfamily of pentameric cys-loop receptors. It has been cloned one beta and four alpha subunits of glycine receptor, which are specifically distributed in different areas of the nervous system. Due to their specific molecular properties and distribution, different subunits ensure important physiological functions: from control of motor activity and regulation of neuronal differentiation to sensory information processing and modulation of pain sensitivity. In this review we briefly describe main functions of these transmembrane proteins, their distribution and molecular architecture. Special attention is paid to recent studies on the molecular physiology of these receptors, as well as on presenting of molecular domains responsible for their modulation and dysfunction.

The functions of the evolutionary conservative complex NAC (Nascent polypepetide Associated Complex) and its subunits are discussed. The heterodimeric NAC protein contains alpha- and beta-subunits and is found to be reversibly bounded to the ribosome in all eukaryotes, from yeast to humans. NAC contacts the nascent polypeptide and protects it from proteolysis. NAC participates in polypeptide chain folding and modulates protein secretion and transmembrane protein formation. Mutations and deletions of genes, encoding NAC subunits are lethal in early development of multicellular eukaryotes. NAC is involved in the ribosome biogenesis. The beta-subunit interacts with caspase-3 and may be involved in the regulation of the apoptotic pathway. The variants of NAC proteins can be considered as chaperone complexes, involved in the response of the cell and the organism to stress factors, as well as regulators of apoptosis. The genes encoding beta-subunits are rapidly evolved, their duplications cause the formation of tissue specific beta-subunit variants with a different number of putative caspase cleavage sites. The homodimer of alpha-subunits is shown to be the RNA/DNA binding protein and acts as a transcriptional cofactor. The diversity in the functioning of NAC is a prime example of a protein that performs a variety of biological functions (moonlighting protein).

The structural organization of inulinases from yeasts, fungi, and plants are researched. For studying their sizes, molecular weight, and permolecular organization, an approach consisting of a combination of atomic force microscopy with methods of dynamic light scattering, gel chromatography, and electrophoresis was used. It is shown that inulinases from Kluyveromyces marxianus and Aspergillus niger form geterodimers and inulinases from tubers of Helianthus tuberosus are present as both dimers and monomers. The role of various forms in the functional activity of inulinase molecules is discussed.

The Ocr protein, encoded by 0.3 (ocr) gene of bacteriophage T7, belongs to the family of antirestriction proteins that specifically inhibit the type I restriction-modification systems. Native Ocr forms homodimer (Ocr)2 both in solution and in the crystalline state. The Ocr protein belongs to the family of mimicry proteins. F53D A57E and E53R V77D mutant proteins were obtained, which form monomers. It was shown that the values of the dissociation constants Kd for Ocr, Ocr F53D A57E and Ocr F53RV77D proteins with EcoKI enzyme differ in 1000 times: Kd (Ocr) = 10(-10) M, Kd (Ocr F53D A57E and Ocr F53R V77D) = 10(-7) M. Antimodification activity of the Ocr monomeric forms is significantly reduced. We have shown, that Ocr dimeric form has fundamental importance for high inhibitory activity.

With a number of experimental methods (circular dichroism, viscosity, intrinsic fluorescence and fluorescence labelling) the conformational folding-unfolding transitions in a compact monomeric form of the Caf1(13-149) subunit were studied under the action of guanidine hydrochloride in the temperature range from 5 to 45 degrees C. It has been shown that transitions always occur between two major states (unfolded and compact). It has made it possible to determine all main thermodynamic functions that characterize the compact state of the Caf1(13-149) subunit: temperature stability T(m), the free energy of stabilization deltaG(st), enthalpy deltaH(tr) and heat capacity jump deltaC collapse of the structure. Data obtained have been confirmed by an independent experiment on melting of fluorescently labeled proteins.

Enzyme AMPK is a part of the family of serine/threonine specific protein kinases. AMPK plays important role in the transfer extracellular signals through phosphorylation of multiple substrates in different metabolic reactions of skeletal muscles. AMPK is geterotrimetric complex, consisting of the catalytic subunit (AMPKalpha) and two regulatory subunits (AMPKbeta and AMPKgamma), which are encoded by seven different high-homologous genes (alpha1, alpha2, beta1, beta2, gamma1, gamma2, gamma3). AMPK regulates skeletal muscle metabolism through phosphorylation of various enzymes such as carbohydrate, lipid and protein metabolism, as well as factors of transcription and initiation. The AMPK expression occurs in response to a changing metabolic requests muscle cells and it leads to increased energy metabolism. The data of recent studies suggest the important role of AMPK in the regulation of intracellular metabolism and point to the need to study the molecular mechanisms involved in the regulation of gene expression in skeletal muscle.

The transcription factor ThrA, which belongs to the MerR transcription regulators, in Bacillus subtilis cells controls genes of nitrogen metabolism under conditions of nitrogen limitation. As all the DNA-binding proteins, it is present as a dimer in cells, but the dimerization site is still unknown. The multiple alignment of TnrA homologs from the other Bacilli allowed to identify the putative dimerization sites. Using the C-terminal truncated TnrA proteins it is established, that, in contrast to other MerR-proteins, the TnrA C-terminus does not participate in dimerization. The surface plasmon resonance has revealed that C-terminus truncations of TnrA do not inactivate its DNA-binding activity. By contrary, it increased an affinity to DNA, confirming that C-terminus controls the DNA-binding activity in a full-length TnrA.

Enzymatic preparations of two isoforms of succinate dehydrogenase (SDG) with specific activity of 22.00 E/mg of protein were obtained from the colorless sulfur bacterium Sphaerotilus natans D-507 cultured organotrophically. Both SDG forms were shown to be heteromers with subunit molecular masses of 70.8, 35.0, 31.8, and 16.2 kDa. The K(m) values for the first and the second forms of SDG were evaluated as 0.615 and 0.531 mM, respectively, with an optimal pH value of 7.2. It was found that the Cl- ion has an activating effect on the SDG activity that can be explained by the specific chemical modification of the enzyme molecule. The results suggest that the isolated enzyme forms are included in different multienzyme complexes, which provide the functioning of the tricarboxylic acid cycle, and SDG preparations can be used for the investigation of other enzyme systems or in vitro modeling of supramolecular cellular structures.

Interrelationship between total, multimer (MM) and oligomer (OM) adiponectin blood concentrations with some metabolic parameters has been investigated in 49 men and 49 women (mean age of 57.3 +/- 10.1 years). We have found negative correlations between total blood adiponectin and its MM form content with body mass index, waist circumference, the insulin resistance index HOMA, with blood concentrations of insulin, glucose, free fatty acids, triglycerides and also positive correlations with high density lipoprotein cholesterol content. There was a poor correlation between OM adiponectin concentration with any parameter studied. According to the regression analysis, concentration of total adiponectin but not its MM form was an independent determinant of the HOMA index in women and free fatty acid concentration in men. In the group of men with the low level of adiponectin its MM form but not total adiponectin reversely correlated with the HOMA index and was its independent determinant. Thus, correlation between blood adiponectin concentration and metabolic parameters is associated with its MM rather than OM form. Study of the role of adiponectin in development of metabolic disorders may be limited to determination of total blood adiponectin concentration except a group of male patients characterized by a low level of this adipokine. In these patients concentrations of the MM form should be determined.

Ability of site-specific nickase BspD6I (Nt.BspD6I) to oligomerize at concentrations > or = 0.5 microM (> or = 0.035 mg/mL) is studied. Three states of Nt.BspD6I are registered via electrophoretic studies both in the presence and in the absence of DNA. Estimation of their molecular mass allows assigning them as a monomer, a dimer and a trimer. Both dimeric and monomeric Nt.BspD6I are shown to hydrolyze its DNA substrate with the identical specificity. Calculation of the electrostatic potential distribution on the Nt.BspD6I globule surface shows that the protein molecule is a dipole. The Nt. BspD6I oligomeric forms are likely to be the result of ionic protein interactions.

Colchicin, podophylotoxin and indibulin are natural cytostatics that are used in the treatment of neoplasms. But applications of those compounds are rather restricted due to the high toxicity and low specificity. It seems very promising to investigate possibility to design new analogs of the above mentioned drugs that will possess higher cytostatic activity and less toxicity. For this purpose we see computer modeling experiments of tubulin and above mentioned compounds interaction as a powerful approach to design new artificial cytostatics with desired properties. In the current study the CHARMM software of protein-ligand interaction molecular dynamics method has been used. Particularly the following strategy has been applied. Molecules of the cytostatits have been positioned at several random positions around binding sites of tubulin and after energy minimization several binding sites have been identified on the tubulin macromolecule. In these binding sites structural changes that may be responsible for tubulin polymerization have been detected.

This review summarizes current data on structure of the most representative group of the collagen family--fibrillar collagens. Attention has been focused on structural organization of individual domains and their functional role in the hierarchical stacking of alpha-chains of collagens. There is presented characteristics of the main stages of biosynthesis and of supramolecular processing of fibrillar collagens. Also considered are some aspects of evolution of fibrillar collagens. The role of duplication of genome and genes, intergene rearrangements, and exon shuffling in evolution of collagen genes is discussed.

Bacterial L-asparaginases, which are widely used in the antitumor therapy, act only as homotetramers, because their active sites are located at the interface between the subunits of the enzyme. Since salt bridges substantially stabilize L-asparaginase tetramers, we have supposed that oligomerization of bacterial L-asparaginase is a high-avidity process. This assumption was proved by bioinformatic and biosensoric methods. It was shown, that a stable tetrameric complex can be formed only by the subunits of the same L-asparaginase. Using two mutants of L-asparaginase Helicobacter pylori it was shown that specificity of molecular recognition is significantly reduced even by single point mutation at the interface of high-homologous closely-related subunits.

The presence of actin-binding protein, tropomyosin, shaped as particles or protein complexes that have no bonds with actin structures were found while the analisys of structural rearrangements of actin cytoskeleton. However, their functioning is still unknown. To study the composition and properties of these protein complexes a novel method of their separation from the cells without destroying the structures of the cytoskeleton have been developed. The protein composition of isolated tropomyosin particles has been analised by gel filtration, electrophoresis and Western blotting. They appeared to be a multimolecular complexes of about 700 kDa. Beside the tropomyosin and actin these complexes also contain the Hsp70, Hsp90 and myosin-9 identified by mass spectrometry analisys. Also, under inhibition of deacetylases by trichostatin A, changes in the number of particles and redistribution of tropomyosin between cytosol and cytoskeleton take place along with actin cytoskeleton rearrangements. The results obtained give a reason to assume that these multimolecular complexes may participate in the process of reorganization of the actin microfilaments.

Alpha-actinin 4 (ACTN4) belongs to actin binding proteins of the spectrin superfamily. Structural organisation of actin fibres and focal contacts is considered to be its primary function in a cell. Besides that, nucleocytoplasmic shuffling of ACTN4 and its involvement in nuclear processes were demonstrated. Lately, additional isoforms of ACTN4 resulted from an alternative splicing has been described in various cell types and malignant tumours. In this study, we present investigation of a novel ACTN4 isoform of 80 kDa. The isoform was found in human epidermoid carcinoma cells A431, and it was not detected in human skin fibroblasts, normal human keratinocytes and transformed human embryonic cells HEK293T. Analysis of ACTN4 mRNA in A431 cells showed the presence of a splice variant that lacked the exons 2-8. The deleted exons code two calponin homology domains responsible for ACTN4 binding to F-actin. Intracellular distribution of the described ACTN4 isoform (ACTN4ISO) overexpressed in HEK293T cells differed from that of the full size protein. In the cytoplasm, ACTN4ISO was allocated diffusively with no colocalisation with actin cytoskeleton structures. Intranuclear distribution of ACTN4ISO also differed from that of the full size ACTN4. Nevertheless, immunochemical analysis demonstrated possibility of ACTN4ISO to form heterodimers with the full size protein. Additional investigations of novel isoform interactions with ACTN4 protein partners might clarify its functional features in A431 cells.

The three-dimensional structure of the 5-HT3 receptor is currently unknown. An available structure of the nicotinic acetylcholine receptor closely related by homology to the 5-HT3 receptor was used as a template for the computer-based homology modeling of the 5-HT3 receptor. The study of the ion migration through the channel by the stirred molecular dynamics method has shown that the steric factor in the region of residue THR 279 and the region of GLU 272, ASP 293 influences the ion transmission. The characteristic of the close interaction between the ion and the amino acid substitutions of the 5-HT3 channel was studied by computing the energy profile using constraint force molecular dynamic simulations. The amino acid sequence responsible for selective ion transmission has been investigated. The structure of the channel domain of the serotonin 5-HT3 receptor as a universal functional unit of the ligand-gated ion channels was discussed.

It has been shown by classical biophysical and biochemical methods in combination with atomic microscopy that lipase from Rhizopus niveus exists in a water solution as a dimer with a molecular weight of 96 kDa. The rate of splitting of triglycerides by a dimeric molecules is twice that of monomers. The heat stability of the monomeric form of lipase at temperatures of 20-60 degrees C is significantly higher than that of the native molecule.

Available data on structure, localization, physiology and pharmacology of GABAB-type receptors are reviewed. GABAB-receptors are pre- and postsynaptically located, belong to metabotropic ones and are connected to trimeric G-protein. Mammal GABAB-receptors, consisting of two subunits of B1 and B2 type, are allosterically regulated heterodimers (actively functioning GABAB-receptors). The activation of GABAB-type receptors leads to increasing K+ release from the cell and hyperpolarization of cell membrane. GABAB-receptors are also connected with potentially dependent Ca2+ channels, which are involved in the synaptic release of neurotransmitters. There is a great number of their allosteric modulators, agonists and antagonists.

The key stage of the infection of the Escherichia coli cell with bacteriophage T4, the binding to the surface of the host cell, is determined by the specificity of the long tail fiber proteins of the phage, in particular, gp37. The assembly and oligomerization of this protein under natural conditions requires the participation of at least two additional protein factors, gp57A and gp38, which strongly hinders the production of the recombinant form of gp37. To overcome this problem, a modern protein engineering strategy was used, which involves the construction of a chimeric protein containing a carrier protein that drives the correct folding of the target protein. For this purpose, the trimeric beta-helical domain of another protein of phage T4, gp5, was used. It was shown that this domain, represented as a rigid trimeric polypeptide prism, has properties favorable for use as a protein carrier. A fragment of protein gp37 containing five pentapeptides repeats, Gly-X-His-X-His, which determine the binding to the receptors on the bacterial cell surface, was fused in a continuous reading frame to the C-terminus of the domain of gp5. The resulting chimeric protein forms a trimer that has the native conformation of gp37 and exhibits biological activity.

Alpha-Crystallin type heat shock protein (alpha-HSP) IbpA from Acholeplasma laidlawii was expressed in Escherichia coil and isolated from cell extract on Ni-sepharose column. Recombinant IbpA, like other alpha-HSPs, spontaneously formed oligomeres in vitro. High resolution electron microscopy revealed regular structures with 15 nm in diameter. Evaluation of molecular mass of IbpA oligomers was performed by gel filtration. Most of oligomers consist of 24 subunits. Recombinant IbpA prevents heat denaturation of soluble proteins in cell extract of E. coli and displays a mild positive effect on thermotolerance of E. coli cells during severe heat shock. We investigated a localization of IbpA in A. laidlawii cell by immunocytochemistry. We suppose that IbpA may protect various intracellular structures from damage during heat shock.

Chaperones are unique remodeling proteins that participate in a great number of intracellular processes and are involved in the correction of protein structure, the prevention of the aggregation of misfolded proteins, the destruction of protein aggregates, and also the unfolding of native protein targets for their translocation across a membrane. In addition to this, Chaperones assist in the dismantling of active oligomers into inactive unfolded monomers for their subsequent photolytic degradation and the assembly of folded subunits into protein assemblies and specific complexes. Data on the structure and functioning of molecular chaperones from five basic families are summarized in the review.

It has been found using a combination of atomic force microscopy with infrared spectroscopy, gel chromatography, and electrophoresis that inulinase from Kluyveromyces marxianus Y-303 has oligomeric structure, which includes two subunits differing in size, molecular mass, and catalytic activity. It has been shown that the division of the inulinase dimer into monomers leads to an increase in the number of irregular sites by 6% for subunit 1 (54.8 kDa) and by 10% for subunit 2 (8.4 kDa) compared with the native enzyme.

Currently inhibitors of protein-protein interactions are considered as perspective prototypes of new generation of drugs. The most attractive targets for such inhibitors are the oligomeric enzymes which active sites are formed by amino acid residues from different subunits. The classic example of such enzyme is HIV protease (HIVp), active only in the homodimeric form. We have developed a new approach for experimental screening of HIVp dimerization inhibitors. It is based on the original biosensoric test-system for differential analysis of interaction of tested substances with HIVp dimers and monomers. Using this test-system we have analyzed the most perspective candidate substances predicted by method of virtual screening, and some derivatives of glycyrrhizin, triterpenic and steroid glycosides. As a result we found one compound, which mainly interacts with HIVp monomers and inhibits in vitro activity of this enzyme with IC50 of about 10(-6) M.

Heat-induced structural transitions of crystallins in the eye lens tissue have been studied by small-angle X-ray scattering. It was shown that a short-time (approximately 1 min) incubation of the bovine eye lens tissue at a temperature of about 60 degrees C leads to a pronounced shift of the small-angle X-ray diffraction maximum due to the short-range order of alpha-crystallin oligomers. This shift indicates an increase in the molecular mass of alpha-crystallin oligomers. The results are evidence that, in the native surrounding and at the native concentration of alpha-crystallin, heat-induced transition of alpha-crystallin quaternary structure takes place. Earlier, this transition of alpha-crystallin has been observed only in solutions and gels of this protein. The results confirm the identity of alpha-crystallin properties in vitro and in vivo.

Cellular and molecular mechanisms of congenital immunity at different levels are discussed including single cell expression patterns and intracellular localization of individual TLR, the use of adapter molecules for generation of activation signals in response to microbial and non-microbial pathogens, soluble trap receptors, and intracellular negative regulators.

Hybridization of M4- and H4-isoenzymes of lactate dehydrogenase in saline medium was studied during slow freezing down to -9.5; -23 and -30 degrees C. It is established that appearances of the enzyme hybrid forms is associated with free water freezing out and with an increase in the concentration of salts in the solution. No hybridization was observed under fast cooling of the enzyme down to -196 degrees C, under exposition in the concentrated solution of chloride and sodium phosphate without freezing and with freezing in deionized water. The mechanism of low-temperature hybridization of lactate dehydrogenase, based on concentration effects of salts which favour dissociation of a protein molecule into subunits, are under discussion. An assumption is advanced on an intensification of intermolecular interactions in liquid microphases of the frozen solution as a possible reason of the subunits recombination into hybrid isoenzymes.