[Oligomerization of site-specific nicking endonuclease BspD6I at high protein concentrations].

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.

[Effect of single-stranded DNA binding proteins on template/primer-independent DNA synthesis in the presence of nicking endonuclease Nt.BspD6I].

In the presence of the Nt.BspD6I nicking endonuclease DNA polymerase Bst stimulates intensive template/primer-independent DNA synthesis. Template/primer-independent DNA synthesis could be the reason for appearing nonspecific DNA products in many DNA amplification reactions particularly in the reactions with using nicking endonucleases. Search of the modes for inhibition template/primer-independent DNA synthesis becomes an urgent task because of broadening the DNA amplification methods with using nicking endonucleases. We report here that the E. coli single-stranded DNA binding protein has no effect on the template/primer-independent DNA synthesis. In the absence of the nicking endonuclease the single-stranded DNA binding protein encoded by bacteriophage T4 gene 32 completely inhibits template/primer-independent DNA synthesis. This protein does not inhibit synthesis of specific DNA product in the presence of nicking endonuclease but remarkably decreases the amount of nonspecific products.

[Effect of various humidity on local dynamic structure of lysozyme in a spin-labeled tetragonal crystal]. / Lokal'naia dinamicheskaia struktura lizotsima v spin-mechenom tetragonal'nom kristalle pri izmenenii vlazhnosti.

The dynamics of the side groups of amino acid residues and local conformational changes in the lysozyme molecule upon dehydration and rehydration of lysozyme crystals were studied by the methods of spin label, X-ray diffraction, and molecular dynamics. The His15 residue of lysozyme from chicken egg white was modified by spin label, and spin-labeled tetragonal crystals of the protein were grown. The spatial structure of the covalently bound spin label and its immediate surroundings in the lysozyme tetragonal crystal was determined. The conformation of a fragment of the lysozyme molecule with the spin label on His15, optimized by the method of molecular dynamics, closely agreed with X-ray data. It was found by the X-ray diffraction analysis that a decrease in relative humidity to 40% is accompanied by both a decrease in the unit cell volume by 27% and a change in the diffraction field of roentgenograms from 0.23 to 0.60 HM. The dehydration of spin-labeled lysozyme crystals leads to an anomalous widening of EPR peaks without changes in their position. The dehydration in the humidity range studied has a two-stage character. The decrease in humidity to 75% is accompanied by a sharp change in the parameters measured, and on further decrease in humidity to 40% they change insignificantly. The first stage is caused by the removal of the greater part of molecules of bulk water, and the second stage is due to the removal of the remaining bulk water and possible changes in the dynamics of weakly bound water molecules and their position. The simulation of experimental EPR spectra showed that the anomalous broadening of the spectrum upon dehydration is related to an increase in the dispersion of spin label orientations induced by changes in the network of hydrogen bonds generated by water molecules in the vicinity of the spin label and a possible turn (by no more than 5 degrees) of the entire protein molecule. After rehydration, the physical state of the lysozyme crystal did not return to the starting point.

[Dynamic mobility of the histidine-containing domain of spin-labeled lysozyme]. / Dinamicheskaia podvizhnost' gistidinsoderzhashchego domena spin- mechenogo lizotsima.

The hen egg-white lysozyme was modified by the spin label (2,2,6,6-tetramethylpiperidine-N1-oxyl-4-iodacetamide) at the single histidine residue His-15. The rotational correlation time of the molecular carrier was found to be defined by the mobility of the histidine-bearing domain and not influenced by the protein monomer shape at pH 4.7 and dimer shape at pH 7.1. The dependence of viscosity at 1 degree C on the distance between outer wide peaks in the immobilized EPR spectra enabled us to evaluate rotational correlation time of the domain. The molecular mass of the latter was close to the data obtained by X-ray analysis. The spin label was highly mobile at room temperature, as the EPR spectrum displayed the triple shape; at 1 degree C it was immobilized. The new general approach to the EPR spectra simulation was applied to all experimental EPR spectra. This approach is based on a substitution of an undefined stochastic process of the spin label reorientation relative to the lysozyme domain by the defined modelled stochastic processes: axial rotation of the nitroxide relative to the preferable axis and angular oscillations of the nitroxide relative to axes of the molecular coordinate system. Each of the modelled stochastic processes leads to a relative partial averaging of the magnetic tensor components. A set of discrete partially averaged states is introduced with the relative cluster of the spin-labelled molecules. The resulting EPR spectrum is assumed to be the sum of EPR spectra from all the clusters. A good fitting of all simulated EPR spectra is obtained.

[Cross-linking of crystalline sperm whale myoglobin using glutaraldehyde]. / Issledovanie fiksatsii glutarovym al'degidom molekul mioglobina kashalota v kristalle.

The salt composition of the solution has been found, which does not interact with glutaraldehyde and myoglobin at an ionic strength corresponding to that of the mother liquid, i.e., 4.5 M (NH4)2SO4. Cross-linking conditions have been elaborated when crystalline myoglobin retains its native structure: myoglobin in 3 M Cs2SO4 pH 5.4 is cross-linked by diffusion in 5% glutaraldehyde vapours in the same salt solution (5 days, room temperature).

[Optical spectroscopy of methemoglobin from the Lampetra fluviatilis river lamprey and its complexes reduced at 77K]. / Opticheskaia spektroskopiia vosstanovlennykh pri 77K metgemoglobina iz rechnoi minogi Lampetra fluviatilis i ego kompleksov.

The absorption spectra from met-haemoglobin from lamprey Lampetra fluviatilis and its complexes with F-, CN-, N3- and imidazole were studied in non-equilibrium states arising in low-temperature (77K) reduction of water-glycerol of oxidized forms of protein with thermolyzed electrons. The Soret band was shown to be not split in low-temperature absorption spectra of reduced met-haemoglobin and its complexes unlike that of the equilibrium deoxyhaemoglobin. It was concluded that met-haemoglobin has a higher symmetry of the active centre as compared to deoxyhaemoglobin.

[T2 DNA, modified by 2,2,6,6-tetramethyl-4-bromoacetooxypiperidine-i-oxyl as a template for RNA polymerase from E. coli B]. / Izuchenie matrichnykh svoistv T2-DNK, modifitsirovannoi 2,2,6,6,-tetrametil-4-bromatsetoksipiperidin-1--oksilom, v RNK-polimeraznoi sisteme E. coli B.

T2-DNA was modified by 2,2,6,6-tetramethyl-4-bromoacetooxypiperidine-1-oxyl (I) at different NaCl concentrations (10(-1) M NaCl--10(-4) M NaCl). Modified DNA were investigated as templates for the RNA-polymerase from E. coli B. It was shown that T2-DNA modified I in 0,1 M NaCl completely preserves the native secondary structure, has a low degree modification (1 molecule I per 1000-2000 nucleotide pairs), but is a noneffective template for the RNA-polymerase from E. coli B (20%-40% as compared with unmodified T2-DNA). Under these conditions the modification occurs probably at the "weakest" (readily melting) sites of DNA. The role of these "weak" sites on DNA as promotors is discussed. The modification of T2-DNA by reagnet I has a stronger inhibitory effect on the total RNA synthesis than on the RNA-synthesis stable to rifampicin. Possible existence of two kinds of "early" promotors on T2-DNA is assumed.