Synthesis of a tetranitrosyl iron complex with unique structure and properties as an inhibitor of phosphodiesterases.

A novel neutral tetranitrosyl iron complex {[Fe(H2O)4]2+[FeR2(NO)2]22-}·4H2O (1) with R = 5-(3-pyridyl)-4H-1,2,4-triazole-3-thiolyls (C7H5N4S), which is a supramolecular ensemble, has been synthesized and studied. As follows from X-ray diffraction analysis, this is an octahedral Fe2+complex (Lewis acid) with two monoanionic dinitrosyl groups [FeR2(NO)2]- (Lewis base) and 4 water molecules as the ligands. As follows from Mössbauer spectra, the coordinating Fe2+ ion is in a low-spin state S = 0, and the dinitrosyl Fe+ ion is in a low-spin state S = 1/2. According to the data of EPR spectroscopy, mass-spectrometry and amperometry, complex 1 in solution forms dinitrosyl particles of [Fe(C7H6N4S-H)2(NO)2]- composition, which are responsible for NO generation. In addition, complex 1 was shown to be a 5-6 times more efficient phosphodiesterase (PDE) inhibitor at 5 × 10-5 M and 10-4 M concentrations than its thioligand. Probable binding sites of the [FeR2(NO)2]- ligand for the bovine PDE1B model have been determined by molecular docking and quantum-chemical calculations.

Synchrotron single-crystal XRD, IR-, Raman spectroscopy and high pressure study of synthetic krieselite.

Spontaneous crystals of krieselite (Ge analogue of topaz) with the chemical formula Al2[(Ge0.75Si0.25)O4](F1.63OH0.37) were synthesized using a thermo-gradient hydrothermal method at a temperature of 600/650 °C and pressure of 100 MPa. The unit cell parameters are: a = 8.9732(8) Å, b = 8.4823(7) Å, c = 4.7379(5) Å, V = 360.62(6) Å3, space group Pnma. The F-/OH- content of the samples was refined by FTIR spectroscopy method. Raman spectroscopy showed the main differences between the spectra of krieselite and topaz at the ambient conditions. The assignment of observed and calculated Ag bands (cm-1) for non-polarized Raman spectra was carried out. Using in situ Raman spectroscopy at high pressures, the dependence of the shift in the position of the main bands of the krieselite Raman spectrum on the pressure was established, and the corresponding paths of pressure induced distortion of crystal structure was assumed. According to the data of Raman spectroscopy, it was revealed that krieselite does not undergo the phase transitions up to 30 GPa. The probable way of crystal structure distortion within the space group Pnma was proposed based on simulation of high-pressure Raman spectra.

Concerted action of two subunits of the functional dimer of Shewanella oneidensis MR-1 uridine phosphorylase derived from a comparison of the C212S mutant and the wild-type enzyme.

Uridine phosphorylase (UP; EC 2.4.2.3), a key enzyme in the pyrimidine-salvage pathway, catalyzes the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. The structure of the C212S mutant of uridine phosphorylase from the facultatively aerobic Gram-negative γ-proteobacterium Shewanella oneidensis MR-1 (SoUP) was determined at 1.68 Šresolution. A comparison of the structures of the mutant and the wild-type enzyme showed that one dimer in the mutant hexamer differs from all other dimers in the mutant and wild-type SoUP (both in the free form and in complex with uridine). The key difference is the `maximum open' state of one of the subunits comprising this dimer, which has not been observed previously for uridine phosphorylases. Some conformational features of the SoUP dimer that provide access of the substrate into the active site are revealed. The binding of the substrate was shown to require the concerted action of two subunits of the dimer. The changes in the three-dimensional structure induced by the C212S mutation account for the lower affinity of the mutant for inorganic phosphate, while the affinity for uridine remains unchanged.

Incorporation of copper ions into crystals of T2 copper-depleted laccase from Botrytis aclada.

Laccases belong to the class of multicopper oxidases catalyzing the oxidation of phenols accompanied by the reduction of molecular oxygen to water without the formation of hydrogen peroxide. The activity of laccases depends on the number of Cu atoms per enzyme molecule. The structure of type 2 copper-depleted laccase from Botrytis aclada has been solved previously. With the aim of obtaining the structure of the native form of the enzyme, crystals of the depleted laccase were soaked in Cu(+)- and Cu(2+)-containing solutions. Copper ions were found to be incorporated into the active site only when Cu(+) was used. A comparative analysis of the native and depleted forms of the enzymes was performed.

Structure and functional studies of the ribonuclease binase Glu43Ala/Phe81Ala mutant.

Ribonuclease from Bacillus intermedius (binase) is a small basic protein with antitumour activity. The three-dimensional structure of the binase mutant form Glu43Ala/Phe81Ala was determined at 1.98 Å resolution and its functional properties, such as the kinetic parameters characterizing the hydrolysis of polyinosinic acid and cytotoxicity towards Kasumi-1 cells, were investigated. In all crystal structures of binase studied previously the characteristic dimer is present, with the active site of one subunit being blocked owing to interactions within the dimer. In contrast to this, the new mutant form is not dimeric in the crystal. The catalytic efficiency of the mutant form is increased 1.7-fold and its cytotoxic properties are enhanced compared with the wild-type enzyme.

Influence of intermolecular contacts on the structure of recombinant prolidase from Thermococcus sibiricus.

Prolidases are peptidases that are specific for dipeptides with proline as the second residue. The structure of recombinant prolidase from the hyperthermophilic archaeon Thermococcus sibiricus (Tsprol) was determined at 2.6 Šresolution. The homodimer of Tsprol is characterized by a complete lack of interactions between the N- and C-terminal domains of the two subunits and hence can be considered to be the most open structure when compared with previously structurally studied prolidases. This structure exists owing to intermolecular coordination bonds between cadmium ions derived from the crystallization solution and histidine residues of a His tag and aspartate and glutamate residues, which link the dimers to each other. This linking leads to the formation of a crystal with a loose packing of protein molecules and low resistance to mechanical influence and temperature increase.

[A new approach for study of structural and functional properties of proteins with unknown functions].

Selected proteins were produced in Escherichia coli bacterial expression system--three proteins from extremophil bacteria: a putative monooxygenase from Deinococcus radiodurans, a putative nucleotidyltransferase from Thermotoga maritima, a putative oxidoreductase from Exiguobacterium sibiricum; and a shaperon from Homo sapiens DJ-1. The protocol of isolation & purification of recombinant proteins were developed that allowed to obtain expression products with the purity of no less than 96%. Conditions for the crystallization have been selected that allowed a stable growth of crystals. Preliminary x-ray experiments were conducted in order to confirm the quality of produced crystals; the resolution of obtained structural data was from 1.2 to 1.8 angstrom.

Characterization of the size and orientation of Na and Cl(2) nanocrystals in electron irradiated NaCl crystals by means of synchrotron radiation.

Samples of synthetic NaCl crystals have been exposed to doses of electron irradiation up to 10(-2) TGy (1 Trad) at about 100 °C, and studied subsequently at T = 95 K by means of synchrotron radiation (SR). In addition to the earlier established Kurdjumov-Sachs orientation relationship (K-S OR) for Na precipitates, the following OR is revealed between solid chlorine and the host NaCl crystal system: [Formula: see text], [Formula: see text]. The size and shape of the Cl(2) precipitates has been studied as a function of the amount of radiation damage (i.e. the concentrations of Na and Cl(2)).