[Study of conformational mobility of insulin, proinsulin, and insulin-like growth factors].

In this work, by method of molecular dynamics there was performed comparative analysis of conformational mobility of evolutionary related peptides--insulin, proinsulin, IGF1, and IGF2. The highest mobility has been shown to be characteristic of the proinsulin molecule, while the lower mobility--IGF1 and proinsulin. The rotation radius (Rg) of insulin, IGF1, and IGF2 changes insignificantly. The proinsulin Rg decreases with leaving to plateau after 6000 ps. Graphs of the mean square deviations (RMSD) from initial positions for A- and B-domains are practically identical, which indicates integrity of the carcass formed by A- and B-domains. The proinsulin C-domain behaves sufficiently chaotically. The IGF1 and IGF2 C-domains form the ordered structures reminding horseshoe and elongated hairpin. The particularly major contribution to the IGF2 mobility is made by D-domain by remaining practically immobile in IGF1, which can be the cause of the high stability of IGF1. The obtained data can be used at development of novel effective drugs for treatment of diabetes mellitus, based on principle of evolutionary relationship of peptides.

[Multinuclear blue copper-proteins: the evolutionary design].

The review presents both our and literature data of results of studies of pathways of evolution of the so-called multinuclear blue copper-proteins (MBCP) that have the domain organization. The MBCP are widely spread in living nature, they have been revealed in cells of archais, bacteria, and eukaryotes. Included in the MBCP composition are such different by their functions copper-proteins as oxidases, reductase, blood coagulation factors V and VIII. Most likely, MBCP have been originated from the low-molecular protein-precursor similar topologically with blue electron-transporting protein of the type of cupredoxin, as a result of action of various evolutionary mechanisms: amplification of genes, formation of protein structures by different combinations of domains, a change of size of domains, the segment elongation at the expense of the activational domain, formation and loss of various copper-binding centres, variation of amino acid ligands in such centres, the appearance of centres of binding of other proteins, glycosylation, etc.

Initiation of the 3':5'-AMP-induced protein kinase A Iα regulatory subunit conformational transition. Part I. A202 and A326 are critical residues.

Protein-ligand docking and molecular dynamics studies have shown that the key event initiated by 3':5'-AMP binding to the A- and B-domains of protein kinase A Iα regulatory subunit is formation of a hydrogen bond between 3':5'-AMP and A202(A326) (the residue in parentheses being from the B-domain). The A202(A326) amide group movement associated with the bond formation leads to reorganization of the phosphate binding cassette (PBC) (the short 3(10)-helix becomes the long α-helix). This process results in L203(L327) displacement and finally causes hinge (B-helix) rotation. The L203(L327) displacement and packing into the hydrophobic pocket formed by the PBC and ß2ß3-loop also depends on the ß2ß3-loop conformation. The correct conformation is maintained by R, I, E, but not K at position 209(333) of the A- and B-domains. So, the R209K and R333K mutants have problems with reaching B-conformation. The apo-form of the 3':5'-AMP-binding domain also undergoes transition from H- to B-conformation. In this case, the movement of A202(A326) amide group seems to be a result of reorganization of the PBC into a more stable α-helix.

Initiation of the 3':5'-AMP-induced protein kinase A Iα regulatory subunit conformational transition. Part II. Inhibition by Rp-3':5'-AMPS.

Protein-ligand docking and ab initio calculations have shown that the 3':5'-AMP phosphorothioate analog (Rp-3':5'-AMPS) blocks the A326 amide group displacement typical of transition from the H- to B-conformation within the B-domain of protein kinase A Iα R-subunit. This behavior of Rp-3':5'-AMPS leads to the inhibition of initial stages of hydrophobic relay operation. In accordance with the proposed hypothesis, Rp-3':5'-AMPS similarly to 3':5'-AMP forms a hydrogen bond with the amide group of A326; however, the properties of this bond together with the position of the sulfur atom prevent the movement of A326. Finally, the Rp-3':5'-AMPS-bound domain appears to be locked in the H-conformation, which is in agreement with the X-ray data.

[The perspectives of development of molecular diagnostics of allergy in the microarray format].

All over the world, many people suffer of various forms of allergy. The timely and accurate diagnostics of allergic sensitization is able to prevent the complications and to select a proper mode of therapy. Over the last century, were isolated and characterized hundreds of specific allergenic substances, in the form of extract mainly In 1966, the class E immunoglobulins were discovered and their role in the formation of hypersensitivity type 1 was explained. Hence, the application of techniques of specific sensitization diagnostics became possible in vitro. The diagnostic systems based on the principles of immune-enzyme assay on plate. The appearance and rapid development of microarray technology in the end of XX century provided an opportunity to implement mass screening of specimen on hundreds and thousands markers. The given technique seems to be promising for carrying out a voluminous and rapid allergoscreening. A prototype specimen of test system was developed in the form of microarray with 15 immobilized allergens. The 16 samples of blood serum of patients with allergic diseases were analyzed. The findings demonstrated a high correlation with reference allergosorbent test. The analysis mode implies a direct immobilization of allergen on phase. This approach makes the most of standard extracts unsuitable due to the presence of considerable amount of non-allergenic "ballast" proteins. In most cases, certain groups of allergens, as shared proteins, manifest a high cross-reactivity decreasing the diagnostics specificity. The issue of appropriateness and necessity of implementing the refined or artificial allergenic components is to be discussed. The experiment results confirmed the correctness of conclusions done and approaches chosen. The component-enabling diagnostics in the microarray format will make it possible to enhance the specificity and accessibility of analysis which will undoubtedly affect patients' quality of life.

Thermodynamic analysis of protein kinase A Ialpha activation.

Thermodynamic analysis of protein kinase A (PKA) Ialpha activation was performed using Quantum 3.3.0 docking software and a Gaussian 03W quantum mechanical computational package. Expected stacking interactions between adenine of 3':5'-AMP and aromatic moieties of amino acids were taken into account by means of MP2/6-31G(d) IPCM (isodensity polarizable continuum model) computations (epsilon = 4.0). It is demonstrated that thermodynamically favorable agonist-induced PKA Ialpha activation is mediated by two processes. First, 3':5'-AMP binding is accompanied by structural changes leading to a thermodynamically favorable regulatory subunit conformation, which is hardly realized in the absence of the ligand (DeltaG degrees (R) = -23.9 +/- 8.2 kJ/mol). Second, 3':5'-AMP affinity to the regulatory subunit conformation observed after agonist-induced PKA Ialpha activation is higher than that to inactive holoenzyme complex (DeltaG degrees (3':5'-AMP) = -28.1 +/- 9.7 kJ/mol). ATP is capable of docking into the 3':5'-AMP-binding site B of the regulatory subunit complexed with the catalytic one, resulting in inhibition of kinase activation. True constants of 3':5'-AMP binding to PKA Ialpha holoenzyme were found to be 60 and 57 microM for the regulatory subunit domains A and B, respectively. These constants, unlike the binding equilibrium constant determined using established experimental techniques and ranging from 15 nM to 2.9 microM, are proved to be direct measures of 3':5'-AMP-PKA Ialpha binding affinity. Their values are in a reasonable agreement with the changes in 3':5'-AMP concentration in the cell (2-55 microM) and account for PKA Ialpha activation in response to adequate stimuli.

New horizons of adenosinetriphosphate energetics arising from interaction with magnesium cofactor.

MD DFT:B3LYP (6-31G** basis set, T = 310 K) method is used to study interactions [singlet (S) and triplet (T) reaction paths] between adenosinetriphosphate, ATP(4-), and [Mg(H(2)O)(6)](2+) in water environment, modeled with 78 water molecules. Computations reveal the appearance of low and high-energy states (stable, quasi-stable, and unstable), assigned to different spin symmetries. At the initial stage of interaction, ATP donates a part of its negative charge to the Mg complex making the Mg slightly charged. As a result, the original octahedral Mg complex loses two (S state) or four (T state) water molecules. Moving along S or T potential energy surfaces (PESs), Mg(H(2)O)(4 )or Mg(H(2)O)(2) display different ways of complexation with ATP. S path favors the formation of a stable chelate with the O1-O2 fragment of ATP triphosphate tail, whereas T path favors producing a single-bonded complex with the O2. The latter, being unstable, undergoes a further conversion into a spin-separated complex, also unstable, and two metastable S complexes, which finally arise in two stable, low-energy and high-energy, chelates. The spin-separated complex experiences rapid decomposition resulting in the production of a highly reactive adenosinemonophosphate ion-radical *AMP, early observed in the CIDNP experiment (Tulub 2006). Biological consequences of the findings are discussed.

[Proton migration in a stack of tyrosine molecules induced by Mg(2+) ion and electric field. Mathematical model]. / Migratsiia protonov v stopke molekul tirozina pod vozdeistviem iona Mg(2+) i élektricheskogo polia. Matematicheskaia model'.

Quantum chemistry methods (ab initio, RHF + MP2(FULL), 6-31G** basis set) were used to study proton migration in tyrosine stacks mimicking the proton channel in tubulin and other proteins. When bound to guanosine-5'-triphosphate, Mg2+ favors the dissociation of water in its first coordination shell, thus initiating subsequent proton shifts in the tyrosine chain composed of spatially remote tyrosine residues of tubulin. The process appears to be thermodynamically allowed, delta G298 < 0, with a potential barrier along the proton shift of no more than 0.75 kcal/mol. The exposure to external electrical field of low intensity, which simulates the electric properties of tubulin, promotes proton migration over long distances.

Topological nature of the genetic code.

A model for topological coding of proteins is proposed. The model is based on the capacity of hydrogen bonds (property of connectivity) to fix conformations of protein molecules. The protein chain is modeled by an n -arc graph with the following elements: vertices (alpha -carbon atoms), structural edges (peptide bonds) and connectivity edges (virtual edges connecting non-adjacent atoms). It was shown that 64 conformations of the 4-arc graph can be described in the binary system by matrices of six variables which form a supermatrix containing four blocks. On the basis of correspondences between the pairs of variables in matrices and four letters of the genetic code matrices and supermatrix are converted, respectively, into the triplets and the table of the genetic code. An algorithm admitting computer programming is proposed for coding the n -arc graph and protein chain. Connectivity operators (polar amino acids) are assigned to blocks of triplets coding for cyclic conformations (G, A-in the second position), while anti-connectivity operators (non-polar amino acids) correspond to blocks of triplets coding for open conformations (C, U-in the second position). Amino acids coded by triplets differing by the first base have different structures. The third base for C, U and G, A is degenerated. Properties of the real genetic code are in full agreement with the model. The model provides an insight into the topological nature of the genetic code and can be used for development of algorithms for the prediction of the protein structure.

Cisplatin stops tubulin assembly into microtubules. A new insight into the mechanism of antitumor activity of platinum complexes.

Despite numerous studies considering DNA as a primary target of cisplatin attack, this work is the first to show the pure effect of cisplatin on the process of tubulin assembly/disassembly in vitro. When platinated, tubulin does not assemble into microtubules (direct electron microscopic studies). In place of them, highly stable and inert circled rings arise. Such tubulin aggregates are unable to participate in the process of chromosome separation during the mitosis, thus blocking cell division in living cells, which is a direct evidence of cisplatin antitumor activity. Cisplatin attack on tubulin causing blockage of tubulin assembly occurs via a two-step binding to GTP in the GTP center of tubulin ((195)Pt, (31)P NMR studies). The calculated binding rates are close to those reported in cisplatin-DNA interactions. The mechanism of cisplatin attack on tubulin is proposed.

Crystallization of the isobutylphosphocholine-cholesterol-isobutanol (1:3:3) complex and its investigation by X-ray analysis: interaction of phopholipid headgroups with cholesterol.

A crystal complex consisting of the isobutyl analog of phosphatidylcholine (PC) (isobutylphosphocholine), cholesterol, and isobutanol with molecular ratio 1:3:3 was obtained and investigated by means of X-ray analysis. The complex was shown to correspond to the monoclinic system (sp. gr. P2(1)): a=16.994(10), b=11.314(7), c=28.164(15), beta=104.07(3), V=5252.63 A(3), Z=2, D(calc)=1.0273 g/cm(3). The isobutylphosphocholine molecule is the key component of the complex. Pairs of hydrogen bonds are formed between the (-delta)O-P-O(delta-) group of the isobutylphosphocholine molecule and C-OH groups of two cholesterol and two isobutanol molecules. The third molecules of cholesterol and isobutanol are H-bonded with the (-delta)O-P-O(delta-) group of the isobutylphosphocholine molecule via C-OH groups of isobutanol and cholesterol, respectively. The crystal structure is built up by translation of the complex in multiplicate along the two-fold axis in the direction of axis b. It contains bands formed by isobutylphosphocholine molecules alternately changing their direction. They are fixed by virtue of two zones of electrostatic interactions of the type (-delta)O-P-O(delta-)ellipsis(+)N(CH(3))(3) and are more or less parallel to the bc plane. The structure also contains three-layer domains formed by cholesterol molecules perpendicular to isobutylphosphocholine bands. In the direction of the c-axis isobutylphosphocholine bands alternate with the layers of cholesterol molecules herewith reproducing repeated blocks. The obtained structure is compared with that of crystals of phospholipids and cholesterol and its derivatives.

The effect of the chain length of polynucleotides on their binding with platinum complexes.

The effect of the length of polynucleotides on their binding with platinum complexes was studied. The highest reaction rate was observed in the reaction with guanosine-containing polynucleotides, whereas cytidine- and adenosine-containing polynucleotides were much less efficient. The monoaqua-forms of the platinum complexes exhibited the highest reactivity in the interaction with polynucleotides in solution. The mechanism implies the formation of the monodentate complex at the first stage which is transformed into the corresponding bidentate complex of chelate type at the second stage. Increase in the length of the polynucleotide chain was shown to enhance its interaction with the platinum complexes.