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Igor Zhukov

Publications and source records attributed to Igor Zhukov.

12 recordsLinked to original sources
PubMed2006

Random sampling of evolution time space and Fourier transform processing.

Application of Fourier Transform for processing 3D NMR spectra with random sampling of evolution time space is presented. The 2D FT is calculated for pairs of frequencies, instead of conventional sequence of one-dimensional transforms. Signal to noise ratios and linewidths for different random distributions were investigated by simulations and experiments. The experimental examples include 3D HNCA, HNCACB and (15)N-edited NOESY-HSQC spectra of (13)C (15)N labeled ubiquitin sample. Obtained results revealed general applicability of proposed method and the significant improvement of resolution in comparison with conventional spectra recorded in the same time.

Fourier Analysis↗
PubMed2006

Two-dimensional Fourier transform of arbitrarily sampled NMR data sets.

A new procedure for Fourier transform with respect to more than one time variable simultaneously is proposed for NMR data processing. In the case of two-dimensional transform the spectrum is calculated for pairs of frequencies, instead of conventional sequence of one-dimensional transforms. Therefore, it enables one to Fourier transform arbitrarily sampled time domain and thus allows for analysis of high dimensionality spectra acquired in a short time. The proposed method is not limited to radial sampling, it requires only to fulfill the Nyquist theorem considering two or more time domains at the same time. We show the application of new approach to the 3D HNCO spectrum acquired for protein sample with radial and spiral time domain sampling.

Algorithms↗
PubMed2005

A protein backbone psi and phi angle dependence of 2J(N(i),C alpha(i-1)): the new NMR experiment and quantum chemical calculations.

A new pulse sequence exploiting double- and zero-quantum evolution of two-spin 15N-13C' coherence is proposed for the accurate measurements of 2J(N(i),C alpha(i-1)) coupling constants. Application of the new experiment is presented for 13C,15N-labeled ubiquitin sample. The density functional theory calculations of 2J(N(i),C alpha(i-1)) coupling constants have been performed to study their dependence on both psi(i - 1) and phi(i - 1) angle in model peptides, and the results exhibit a good correlation with experimental data.

Models, Molecular↗
PubMed2005

Apoptosis induced by membrane damage in human lymphocytes; effects of arachidonic acid and its photoproducts.

The effect of arachidonic acid (AA) combined with UVA irradiation was studied in a model system mimicking phototherapy PUVA (psoralen+UVA) ex vivo in vitro. The contribution of damage to the plasma membrane by PUVA was tested on human lymphocytes derived from healthy donors. The effect of arachidonic acid (AA) combined with UVA irradiation was compared with that of a psoralen photoadduct to AA added to the culture. The adduct, obtained photochemically and purified, was characterized by NMR and MS spectrometry as a cycloadduct of psoralen to the vinylene bond of the acid (AA<>PSO). The reactions of cultured cells, manifested 20 h after treatment by changes in apoptosis and mitochondrial depolarization, were monitored by flow cytometry by tagging lymphocytes with appropriate fluorescent probes. Treatment of lymphocyte suspension within AA doses from 40 to 100 microM gradually induced a shift from Anx-V(+) (single positive cells) to late apoptotic, Anx-V(+)PI(+) (double positive cells) in a dose dependent manner. The adduct, AAPSO, induced apoptotic changes at a concentration 2-3 times higher than free AA. Combination of psoralen (1 microM ) or arachidonic acid (20-120 microM) with UVA irradiation (2-6 J/cm(2)) accelerated the plasma membrane changes in a synergic way. Preliminary studies indicated that changes in the transmembrane potential of mitochondria paralleled the apoptosis when cells were treated by AA alone. Our findings showed that UVA radiation of lymphocytes in the presence of arachidonic acid, as in the presence of psoralen, enhanced apoptosis of cells in a synergic manner. Thus, PUVA-induced apoptosis may proceed in part by a still undefined signaling pathway(s) triggered in lymphocyte membranes.

Apoptosis↗
PubMed2004

Redox modifications of the C-terminal cysteine residue cause structural changes in S100A1 and S100B proteins.

S100 is a family of small, acidic, calcium binding proteins involved in the control of a multitude of intra- and extracellular processes, including many pathologies. The application of the analytical methodology based on the combination of RP HPLC and ESI-MS allowed for the characterization of S-nitrosylation and S-glutathionylation in two representative S100 proteins: S100A1 and S100B. The GSNO related S-nitrosylation of the conserved C-terminal cysteine is strongly activated by the binding of Ca(II) to S100A1 and of Ca(II) and Zn(II) to S100B. This modification results in a global alteration of protein structure, as demonstrated by a variety of techniques. The presented results provide a mechanistic basis for further studies of the function of S100 proteins in the control of redox-based and metal-based signal transduction.

Animals↗
PubMed2004

The DQ-HN[CACB] and DQ-HN(CO)[CACB] sequences with evolution of double quantum Calpha-Cbeta coherences.

The new variant of known HNCACB and HN(CO)CACB techniques is proposed that employs excitation and evolution of double quantum Calpha-Cbeta coherences. The most important features of the new method are: increased signal dispersion, lack of splittings due to 1J(Calpha-Cbeta) spin-spin couplings, and absence of accidental cancellations of positive and negative signals. The acquisition of both DQ-HN[CACB] and DQ-HN(CO)[CACB] techniques enables sequential assignment of protein backbone, using only Calpha-Cbeta DQ-frequencies. The determination of all Calpha and Cbeta chemical shifts requires, however, a comparison with HN(CO)CA or HNCA spectra. Examples of applications of the DQ-HN[CACB] and DQ-HN(CO)[CACB] experiments are presented, employing the 2D Reduced Dimensionality approach for 13C, 15N-labeled ubiquitin, and the 3D acquisition for 13C, 15N-double labeled Ca2+ -binding bovine S100A1 protein in the apo state (21 kDa) with overall correlation time of 8.1 ns.

Animals↗
PubMed2004

The set of triple-resonance sequences with a multiple quantum coherence evolution period.

The new pulse sequence building block that relies on evolution of heteronuclear multiple quantum coherences is proposed. The particular chemical shifts are obtained in multiple quadrature, using linear combinations of frequencies taken from spectra measured at different quantum levels. The pulse sequences designed in this way consist of small number of RF-pulses, are as short as possible, and could be applied for determination of coupling constants. The examples presented involve 2D correlations HNCO, HNCA, HN(CO)CA, and H(N)COCA via heteronuclear zero and double coherences, as well as 2D HNCOCA technique with simultaneous evolution of triple and three distinct single quantum coherences. Applications of the new sequences are presented for 13C,15N-labeled ubiquitin.

Carbon Isotopes↗
PubMed2003

Characterization of the low pH solution structure and dynamics of the region 4 of Escherichia coli RNA polymerase sigma70 subunit.

Solution structure of the region 4 of sigma(70) subunit of Escherichia coli RNA polymerase, whose 4.2 subregion is involved in specific recognition of the -35 element of cognate promoters, has not been yet studied. Using multinuclear NMR spectroscopy, we have assigned recently all the backbone and aliphatic side-chain (13)C resonances for a recombinant His(6)-tagged protein containing the whole region 4 and a part of region 3.2 of sigma(70) in aqueous solution at pH 2.8 (Poznański, J., Zhukov, I., Bolewska, K., and Wierzchowski, K. L. (2001) J. Biomol. NMR 20, 181-2). The protein proved to be sufficiently soluble and did not aggregate only in the protonated state. In this paper, the structure and dynamics of this state at pH 2.8 have been extensively examined using CD and NMR spectroscopy. Both analysis of CD spectra and NMR observables (secondary chemical shifts of the (13)Calpha, (13)CO, and (1)Halpha nuclei and of vicinal (3)J(HNH)(alpha) coupling constants) indicated that a significant amount of helical structure remained in the protonated protein. The amount of this structure increased upon deprotonation of carboxylic amino acids, as shown by pH titration CD experiments. 2,2,2-Trifluoroethanol induced an even more extensive build up of this structure. Distribution along the protein sequence of the secondary shifts and (3)J(HNH)(alpha) couplings demonstrated partition of the helical secondary structure into three helices located similarly as in the crystal structures of the homologous region 4 of the sigma(A) subunit of Thermus aquaticus RNA polymerase (Campbell, E. A., Muzzin, O., Chlenov, M., Sun, J. L., Olson, A., Weinman, O., Trester-Zedlitz, M. L., and Darst, S. A. (2002) Mol. Cell 9, 527-39) and sigma(70) of the Thermus thermophilus RNA polymerase (Vassylyev, D. G., Sekine, S., Laptenko, O., Lee, J., Vassylyeva, M. N., Borukhov, S., and Yokoyama, S. (2002) Nature 417, 712-9.). Spectral density analysis of NMR relaxation parameters, R(1) and R(2), and [(1)H]-(15)N heteronuclear NOEs indicated that backbone fluctuations in the whole region embracing the three helices and intervening nonhelical sequences are severely restricted on the nanosecond time scale as compared with the N- and C-terminal protein segments. Inspection of the side-chain contacts stabilizing the crystal structures well explains the observed folding and solution properties of sigma(70)(4) protein in its protonated state.

Amino Acid Sequence↗
PubMed2003

Multiple quadrature detection in reduced dimensionality experiments.

A new, simple procedure is proposed which enables acquisition of two or more chemical shifts encoded in a common dimension simultaneously in quadrature. For n chemical shifts projected in a single dimension, the expected effect is obtained by interleaved acquisition and appropriate combination of 2(n) data sets per increment of respective evolution time. The particular chemical shifts can be calculated from sums and differences of signal frequencies obtained by different combination of the acquired data sets. In comparison to the established reduced dimensionality (RD) techniques, the proposed method enhances resolution due to reduction of the number of signals and requires less evolution time increments owing to narrower spectral width in the RD-domain. We show examples of the application of the new approach to the 2D HNCA and HN(CO)CA techniques with two, and 2D HACANH with three frequencies simultaneously encoded in the t(1) evolution period, for (13)C,(15)N-labeled ubiquitin.

Amino Acid Sequence↗
PubMed2002

Conformational solution studies of (Sar7)desamino- and (MeAla7)desamino-vasopressin analogues using NMR spectroscopy.

Solution structures of two analogues of vasopressin with an amino acid sequence of c[Mpa1-Tyr2-Phe3-Gln4-Asn5-Cys6]-Xaa7-Arg8-Gly9-NH2 (Xaa = Sar [I] or MeAla [II]) were established using ROE and the 3J(HNH alpha) couplings. Each of the peptides was found to exist in two stable isomers, pertaining to the cis or trans status of the Cys6-Xaa7 peptide bond, thus giving rise to four study objects. Two methods for studies of the conformational properties of the structures were compared. In the first, the algorithm consisted of three steps: (i) An Electrostatically Driven Monte-Carlo (EDMC) search for low-energy conformations. (ii) Simulations of the NOESY spectra and the vicinal couplings for these conformations. (iii) Determination of the statistical weights of the conformations with the ANALYZE package, so as to meet the best fit of the averaged NOE intensities and couplings to the experimental data. In the second method, the distance constraints and the torsion angles were used as the usual constraints in the Distance Geometry and Simulated Annealing algorithms. The flexibility of the pressin ring and the C-terminus was characterized by a large number of families of conformations. The presence of the beta-turn at position 4,5 was confirmed for all low energy structures found. The use of the EDMC method for the elaboration of the NMR data for small flexible peptides yielded an adequate description of their conformational diversity and is the method of choice for the analysis of their solution structures.

Algorithms↗
PubMed2002

Hydrogen bonds in human ubiquitin reflected in temperature coefficients of amide protons.

Analysis of amide proton temperature coefficients (deltasigma(HN)/DeltaT) in human ubiquitin shows their usefulness in indicating hydrogen bonds. The availability of a very accurate solution structure of ubiquitin enables the precise determination of hydrogen bonds and increases the reliability of the analysis of chemical shift temperature gradients. Values of deltasigma(HN)/DeltaT more positive than -4.6 ppb/K are very good indicators of hydrogen bonds. Additionally, a weak temperature dependence of non-hydrogen-bonded amides was observed for amide protons that are significantly shifted upfield. We observed that temperature gradients of amide protons involved in short hydrogen bonds are related to donor-acceptor distances.

Amides↗