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Biomedical subjects

A Volkov

Publications and source records attributed to A Volkov.

At least 19 recordsLinked to original sources
PubMed2003

A prokaryotic condensin/cohesin-like complex can actively compact chromosomes from a single position on the nucleoid and binds to DNA as a ring-like structure.

We show that Bacillus subtilis SMC (structural maintenance of chromosome protein) localizes to discrete foci in a cell cycle-dependent manner. Early in the cell cycle, SMC moves from the middle of the cell toward opposite cell poles in a rapid and dynamic manner and appears to interact with different regions on the chromosomes during the cell cycle. SMC colocalizes with its interacting partners, ScpA and ScpB, and the specific localization of SMC depends on both Scp proteins, showing that all three components of the SMC complex are required for proper localization. Cytological and biochemical experiments showed that dimeric ScpB stabilized the binding of ScpA to the SMC head domains. Purified SMC showed nonspecific binding to double-stranded DNA, independent of Scp proteins or ATP, and was retained on DNA after binding to closed DNA but not to linear DNA. The SMC head domains and hinge region did not show strong DNA binding activity, suggesting that the coiled-coil regions in SMC mediate an association with DNA and that SMC binds to DNA as a ring-like structure. The overproduction of SMC resulted in global chromosome compaction, while SMC was largely retained in bipolar foci, suggesting that the SMC complex forms condensation centers that actively affect global chromosome compaction from a defined position on the nucleoid.

Adenosine Triphosphate↗
PubMed2001

Critical examination of the radial functions in the Hansen-Coppens multipole model through topological analysis of primary and refined theoretical densities.

A double-zeta (DZ) multipolar model has been applied to theoretical structure factors of four organic molecular crystals as a test of the ability of the multipole model to faithfully retrieve a theoretical charge density. The DZ model leads to significant improvement in the agreement with the theoretical charge density along the covalent bonds and its topological parameters, and eliminates some of the bias introduced by the limited flexibility of the radial functions when a theoretical density is projected into the conventional multipole formalism. The DZ model may be too detailed for analysis of experimental data sets of the accuracy and resolution typically achieved at present, but provides guidance for the type of algorithms to be adapted in future studies.

Journal Article↗
PubMed2001

Density-optimized radial exponents for X-ray charge-density refinement from ab initio crystal calculations.

Structure factors based on periodic density-functional (DFT) calculations on 25 molecular crystals have been used to evaluate trends in refined values of the kappa and kappa' expansion-contraction parameters of the Hansen-Coppens multipole formalism. As found previously and expected physically, the spherical-valence-shell kappa parameters are closely related to the net atomic charges, negative atoms being expanded and vice versa. kappa' parameters, which scale the radial dependence of the non-spherical deformation functions, are remarkably consistent for particular bonding environments. Systematic trends are observed for both carbon and oxygen, but the values obtained for nitrogen show a larger variation. Average values for oxygen and carbon in different bonding environments are tabulated and can be used whenever refinement of experimental data is affected by lack of uniqueness of the charge-density parameter set. Values for nitrogen must be more finely tuned to the specific bonding environment. The relation between atomic charge and kappa offers the possibility of introducing a constraint in the charge-density refinement of very large molecules, for which reduction of the size of the parameter set may be essential.

Journal Article↗
PubMed2000

Evaluation of net atomic charges and atomic and molecular electrostatic moments through topological analysis of the experimental charge density

The atoms in molecules (AIM) theory may be used to derive atomic charges, atomic volumes and molecular dipole moments from the charge density. The theory is applied to theoretical periodic Hartree-Fock (PHF), density-functional (DFT) and experimental X-ray densities of p-nitroaniline using the program TOPOND and a newly developed program, TOPXD, for topological analysis of densities described by the Coppens-Hansen multipole formalism. Results show that, like dipole moments derived directly from the multipole refinement, AIM-derived atomic and molecular moments are dependent on the multipole model used. As expected, large differences are found between charges derived from the monopole parameters and those from AIM analysis of the experimental model density. Differences between the k'-restricted multipole model (KRMM) and the unrestricted multipole model (UMM) results are preserved in the AIM analysis. The enhancement of the molecular dipole moment of p-nitroaniline in the solid state is confirmed by both experiment and theory but the experimental dipole moment is in much better agreement with theoretical periodic Hartree-Fock and, especially, periodic DFT (PDFT) data when KRMM is used in the refinement. The AIM analysis allows a rigorous definition of the charges of the atoms in molecules and provides a realistic basis for comparison between molecules and between experiment and theory.

Journal Article↗
PubMed2000

On the origin of topological differences between experimental and theoretical crystal charge densities

Topological analysis of experimental and theoretical (molecular and crystal) electron densities of p-nitroaniline and p-amino-p'-nitrobiphenyl reveals considerable discrepancies between experiment and theory for the bond critical points properties. Particularly large differences occur for the positive curvature along the bond path (lambda 3). The differences become somewhat smaller when more extended basis sets and correlation effects are introduced in the theoretical calculations. The effect of the crystal matrix on the properties of bond critical points is evaluated for the p-nitroaniline molecule using the 6-21G** and 6-31G** basis sets. The differences between the isolated molecule and the molecule in the crystal are too small to explain the quantitative disagreement between the theoretical and experimental topologies reported in the literature and found in the current study. For most bonds, the observed changes in the properties of the electron density agree well for both basis sets but some discrepancies are found for changes in lambda 3 for N-H and aromatic C-C bonds. When the theoretical densities are projected into the multipole density functions through refinement of the theoretical structure factors, the topological properties change and differences between theory and experiment are reduced. The main origin of the observed discrepancies is attributed to the nature of the radial functions in the experimental multipole model.

Journal Article↗
PubMed2000

The experimental charge-density approach in the evaluation of intermolecular interactions. Application of a new module of the XD programming package to several solids including a pentapeptide.

A new module interfaced to the XD programming package has been used in the evaluation of intermolecular interactions and lattice energies of the crystals of p-nitroaniline, L-asparagine monohydrate and the pentapeptide Boc-Gln-D-Iva-Hyp-Ala-Phol (Boc = butoxycarbonyl, Iva = isovaline = ethylalanine, Phol = phenylalaninol). The electrostatic interactions are evaluated with the atom-centered distributed multipoles from KRMM (kappa'-restricted multipole model) refinements, using the Buckingham expression for non-overlapping charge densities. Results for p-nitroaniline are compared with Hartree-Fock (HF), density functional (DFT) and Moller-Plesset (MP2) supermolecular calculations and with HF and DFT periodic calculations. The HF and DFT methods fail to predict the stability of the p-nitroaniline crystal but the results of the experimental charge-density approach (ECDA) are in good agreement with both MP2 interaction energies and the experimental lattice energy. ECDA results for L-asparagine monohydrate compare well with those from DFT supermolecular and periodic HF calculations. The disorder of the terminal group in the pentapeptide, which persists at the experimental temperature of 20 K, corresponds to an energy difference of only 0.35 kJ mol(-1), which is too small to be reproduced with current methods.

Aniline Compounds↗
PubMed1999

On the errors in molecular dipole moments derived from accurate diffraction data.

The error in the molecular dipole moment as derived from accurate X-ray diffraction data is shown to be origin dependent in the general case. It is independent of the choice of origin if an electroneutrality constraint is introduced, even when additional constraints are applied to the monopole populations. If a constraint is not applied to individual moieties, as is appropriate for multicomponent crystals or crystals containing molecular ions, the geometric center of the entity considered is a suitable choice of origin for the error treatment.

Journal Article↗
PubMed1998

Systematic study of the six cysteines of the E1 subunit of the pyruvate dehydrogenase multienzyme complex from Escherichia coli: none is essential for activity.

Variants of the Escherichia coli 1-lip pyruvate dehydrogenase multienzyme complex (1-lip PDHc) with the C259N and C259S substitutions in the putative thiamin diphosphate-(ThDP-) binding motif of the pyruvate dehydrogenase component (E1, EC 1.2.4.1) were characterized. Single substitutions were made at the five remaining cysteines of the E1 component, creating the C120A, C575A, C610A, C654A, and C770S variants to test the hypothesis that the activity loss that accompanies exposure of the enzyme to fluoropyruvate, bromopyruvate, and 2-oxo-3-butynoic acid is the result of the modification of approximately one cysteine residue per E1 monomer. Surprisingly, all single cysteine E1 variants could be reconstituted with E2-E3 subcomplex and showed PDHc activity ranging from 74% to 96% that of the parental enzyme. The specific activities of C259N and C259S variants of 1-lip PDHc were 58% and 27% relative to that of the parental 1-lip PDHc. All five single cysteine E1 variants, along with the C259N and C259S variants of 1-lip PDHc, could also (1) be inactivated with fluoropyruvate and 2-oxo-3-butynoic acid, (2) were subject to inactivation by the monoclonal antibody 18A9 reported from one of our laboratories, and (3) were subject to regulation by pyruvate and acetyl-CoA. It was therefore concluded that none of the six cysteine residues is essential for the activity of the E1 component or of the complex. When tested with the putative transition-state analogue, thiamin 2-thiothiazolone diphosphate, all but the C259S and C259N variants were very potently inhibited, the stoichiometry for parental E1 being about 1.6 mol of inhibitor/mol of E1 subunit. The C259S and C259N E1 variants required at least 25-fold greater inhibitor concentration to achieve the same level of inhibition. C259 is located in the putative thiamin diphosphate-binding motif of the enzyme [more exactly, it is adjacent to a ligand to the Mg(II) ion]. It is therefore concluded that thiamin 2-thiothiazolone diphosphate is not a transition-state analogue; rather, it is a potent inhibitor of the complex because of a specific interaction with the C259 residue.

Acetyl Coenzyme A↗
PubMed1996

Evidence for intramolecular processing of prosubtilisin sequestered on a solid support.

Subtilisin E is synthesized in Bacillus subtilis as a preprosubtilisin. The prepeptide is removed by a signal peptidase, and the propeptide is cleaved from the mature protein by the catalytic domain of subtilisin itself in an autocatalytic fashion. A six residue histidine-tag was attached to the C terminus of prosubtilisin and mature subtilisin to enable immobilization on a metal chelating resin. Guanidine-HC1 denatured histidine-tagged subtilisin and prosubtilisin were immobilized on Co2+ charged Talon resin, then renatured by dialysis of the resin against renaturation buffer. Refolding of the immobilized prosubtilisin resulted in its quantitative autoprocessing and the formation of active enzyme. Mature subtilisin on the other hand refolded into an active conformation with very low efficiency, and at the same concentration the steady-state rate attained was at least a 1000 times lower than that from prosubtilisin. The results give very strong support for an intramolecular autoprocessing pathway for prosubtilisin, in addition to an intermolecular one demonstrated before. The results also demonstrate rather convincingly the very much higher yield of active enzyme refolded from prosubtilisin than from mature protein under sequestered unimolecular conditions.

Enzyme Precursors↗
PubMed1996

Three of four cysteines, including that responsible for substrate activation, are ionized at pH 6.0 in yeast pyruvate decarboxylase: evidence from Fourier transform infrared and isoelectric focusing studies.

Oligonucleotide-directed site-specific mutagenesis was carried out on pyruvate decarboxylase (EC 4.1.1.1) from Saccharomyces cerevisiae at three of the four cysteines (152, 221, and 222), the fourth (69) being buried according to X-ray crystallographic results [Arjunan et al. (1996) J. Mol. Biol. 256, 590-600]. All of the variants still retained significant activity, and all could be purified to homogeneity. FT-IR experiments were run on the C221S, C222S, C221S/C222S and C152A variants, as well as on the wild-type enzyme. There is a band present at 2557 cm-1 in the spectra of all variants and the wild-type enzyme, except in the spectrum of the C152A variant. This frequency is appropriate to a cysteine S-H stretching mode. It was therefore concluded that C152 is the only undissociated cysteine on the enzyme at pH 6.0, the pH optimum of this enzyme, whereas C221, C222, and C69 are all ionized. Isoelectric focusing experiments were carried out on all of these variants, as well as on the H92A variant (H92 is across the domain divide on the alpha domain, from C221 located on the beta domain). The variation in isoelectric points deduced from the data was consistent with removal of negative charges concomitant with the C221S, C222S, and C221S/C222S substitutions and removal of a positive charge with the H92A substitution when compared to that of the wild-type enzyme. The results of these two types of experiments are in good accord and suggest that the site of substrate activation at C221 [Baburina et al. (1994) Biochemistry 33, 5630-5635] is comprised of a Cys221S- +HHis92 ion pair, not unlike that found in papain and glyceraldehyde-3-phosphate dehydrogenase. This finding suggests that the regulatory site of this enzyme has been optimized for nucleophilic reactivity between the thiolate of C221 and the keto carbon of the 2-oxoacid.

Cysteine↗
PubMed1985

The noise and functional disturbances of the cardio-vascular system in seamen.

The work presents quantitative relations between service-dependent noise dose levels and "unspecific" for the noise influence remote effects basing on the study of the cardiovascular system of 207 members of the deck and engine room crews. The seamen observed had no hearing losses due to noise (according to the ISO standard 1999). The study carried out in the pause between sea-going voyages showed that when the length of the service exceeded 10 years the noise dose increment of 1 dB corresponds to the hypertension frequency increment of 2%. The increase of the dose and the length of the service turns out as a trend towards the diminished mean arterial pulse pressure (AP) and elevated diastolic and mean dynamic AP. By resorting to the correlation-regression analysis the information value of the mean dynamic AP for the noise influence evaluation was shown as well the possibility to determine the safe noise action dose which may not be over 100 dB at the service length under 40 years.

Adult↗