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A V Kurochkin

Publications and source records attributed to A V Kurochkin.

At least 19 recordsLinked to original sources
PubMed1998

NMR solution structure of the 21 kDa chaperone protein DnaK substrate binding domain: a preview of chaperone-protein interaction.

The solution structure of the 21 kDa substrate-binding domain of the Escherichia coli Hsp70-chaperone protein DnaK (DnaK 386-561) has been determined to a precision of 1.00 A (backbone of the beta-domain) from 1075 experimental restraints obtained from multinuclear, multidimensional NMR experiments. The domain is observed to bind to its own C-terminus and offers a preview of the interaction of this chaperone with other proteins. The bound protein region is tightly held at a single amino acid position (a leucyl residue) that is buried in a deep pocket lined with conserved hydrophobic residues. A second hydrophobic binding site was identified using paramagnetically labeled peptides. It is located in a region close to the N-terminus of the domain and may constitute the allosteric region that links substrate-binding affinity with nucleotide binding in the Hsp70 chaperones.

Amino Acid Sequence↗
PubMed1998

Structural analysis of the P10/11-P12 RNA domain of yeast RNase P RNA and its interaction with magnesium.

The P10/11-P12 RNA domain of yeast RNase P contains several highly conserved nucleotides within a conserved secondary structure. This RNA domain is essential for enzyme function in vivo, where it has a demonstrated role in divalent cation utilization. To better understand the function of this domain, its structure and alterations in response to magnesium have been investigated in vitro. A secondary structure model of the P10/11-P12 RNA domain had been previously developed by phylogenetic analysis. Computer modeling and energy minimization were applied to the Saccharomyces cerevisiae P10/11-P12 domain to explore alternatives and additional interactions not predicted by the phylogenetic consensus. The working secondary structure models were challenged with data obtained from 1H NMR and in vitro chemical and enzymatic probing experiments. The solution structure of the isolated domain was found to conform to the phylogenetic prediction within the context of the holoenzyme. Structure probing data also discriminated among additional base contacts predicted by energy minimization. The withdrawal of magnesium does not appear to cause gross refolding or rearrangement of the RNA domain structure. Instead, subtle changes occur in the solution accessibility of specific nucleotide positions. Most of the conserved nucleotides reported to be involved in magnesium utilization in vivo also display magnesium-dependent changes in vitro.

Base Sequence↗
PubMed1998

High-resolution detection of five frequencies in a single 3D spectrum: HNHCACO--a bidirectional coherence transfer experiment.

A new triple-resonance pulse sequence, 3D HNHCACO, is introduced and discussed, which identifies sequential correlations of the backbone nuclei (H alpha (i-1), C alpha (i-1), C(i-1), NH(i), N(i)) of doubly labeled proteins in H2O. The three-dimensional (3D) method utilizes a recording of 15N and 13C resonances in a single indirect time domain, the 13C' resonance in another indirect time domain, and detects both NH and H alpha protons. A bidirectional coherence transfer (NH(i) <--> N(i) <--> C(i-1) <--> C alpha (i-1) <--> H alpha (i-1)) is effectuated, resulting in a single high-resolution 3D spectrum that contains the frequencies of all five backbone nuclei. The experiment was applied to the 12.3 kDa ribonuclease from Bacillus intermedius (Binase).

Bacillus↗
PubMed1996

[Surgical treatment of children with generalized myasthenia].

The rich experience in the country in surgical treatment of children with generalized myasthenia has been analysed. The comparative analysis of various means of preoperative preparation is done. Stabilisation of myasthenical status is very important in the preoperative preparation, the use of steroid hormones is advocated for this purpose. The indications, contraindications, as well as the scope and optimal timing of surgery are formulated. Two types of clinical and immunomorphological changes are depicted in children with generalized myasthenia that are of certain prognostic value. The rating of risk factors, useful for prognosis of unfavorable results of surgery is proposed.

Adolescent↗
PubMed1995

Occurrence, solution structure and stability of DNA hairpins stabilized by a GA/CG helix unit.

The occurrence and NMR solution structure of a class of biloop hairpins containing the sequence 5'-CGXYAG are presented. These hairpins, which are variations on a sequence found in the reverse transcript of the human T-cell leukemia virus 2 (HLV2), show elevated melting points and high chemical stability toward denaturation by urea. Hairpins with the 5'-CGXYAG configuration have melting points 18-20 degrees higher than hairpins with 5'-CAXYGG or 5'-GGXYAC configurations. The identities of the looping bases, X and Y above, play a negligible role in determining the stability of this DNA hairpin stability. This is very different from G-A based loops in RNA, where the third base must be a purine for high stability [the GNRA loops; V.P. Antao, S.Y. Lai and I. Tinoco, Jr (1991) Nucleic Acids Res., 19, 5901-5905]. We show that these properties are associated with a four base helix unit that contains both a sheared GA base pair and a Watson-Crick CG base pair upon which it is stacked. As an understanding of the significance of AG base pairs has become increasingly important in the structural biology of nucleic acids, we compute an 0.7-0.9 A precision ensemble of NMR solution structures using iterative relaxation matrix methods. Calculations performed on NMR-derived structures indicate that neither base-base electrostatic interactions, nor base-solvent dispersive interactions, are significant factors in determining the observed differences in hairpin stability. Thus the stability of the 5'-CGXYAG configuration would appear to derive from favorable base-base London/van der Waals interactions.

Base Composition↗
PubMed1995

Solution structure of the catalytic domain of human stromelysin complexed with a hydrophobic inhibitor.

Stromelysin, a representative matrix metalloproteinase and target of drug development efforts, plays a prominent role in the pathological proteolysis associated with arthritis and secondarily in that of cancer metastasis and invasion. To provide a structural template to aid the development of therapeutic inhibitors, we have determined a medium-resolution structure of a 20-kDa complex of human stromelysin's catalytic domain with a hydrophobic peptidic inhibitor using multinuclear, multidimensional NMR spectroscopy. This domain of this zinc hydrolase contains a mixed beta-sheet comprising one antiparallel strand and four parallel strands, three helices, and a methionine-containing turn near the catalytic center. The ensemble of 20 structures was calculated using, on average, 8 interresidue NOE restraints per residue for the 166-residue protein fragment complexed with a 4-residue substrate analogue. The mean RMS deviation (RMSD) to the average structure for backbone heavy atoms is 0.91 A and for all heavy atoms is 1.42 A. The structure has good stereochemical properties, including its backbone torsion angles. The beta-sheet and alpha-helices of the catalytic domains of human stromelysin (NMR model) and human fibroblast collagenase (X-ray crystallographic model of Lovejoy B et al., 1994b, Biochemistry 33:8207-8217) superimpose well, having a pairwise RMSD for backbone heavy atoms of 2.28 A when three loop segments are disregarded. The hydroxamate-substituted inhibitor binds across the hydrophobic active site of stromelysin in an extended conformation. The first hydrophobic side chain is deeply buried in the principal S'1 subsite, the second hydrophobic side chain is located on the opposite side of the inhibitor backbone in the hydrophobic S'2 surface subsite, and a third hydrophobic side chain (P'3) lies at the surface.

Amino Acid Sequence↗
PubMed1993

Assignments for the main-chain nuclear magnetic resonances and delineation of the secondary structure of the catalytic domain of human stromelysin-1 as obtained from triple-resonance 3D NMR experiments.

We report the NMR assignments for the main-chain 13C, 15N, and 1H resonances (1HN, 1H alpha, 15N alpha, 13C alpha, 13CO) for the 19.5-kDa catalytic domain of human stromelysin-1, a zinc endoproteinase thought to be involved in pathologic tissue degradation. The assignments were predominantly obtained from triple-resonance three-dimensional NMR experiments using double-labeled (15N/13C) samples. The secondary structure of the molecule was determined from analysis of 3D 15N-resolved NOESY experiments. It was found to consist of a five-stranded mixed beta-sheet with four parallel and one antiparallel strand and three helices. The topological arrangement of the secondary structure elements of stromelysin catalytic domain is remarkably similar to that found for astacin, a Zn proteinase for which the tertiary structure was recently determined from X-ray diffraction data [Bode et al. (1992) Nature 358, 164-167].

Amino Acid Sequence↗
PubMed1993

[Diagnosis, surgical treatment and prognosis in patients with generalized myasthenia].

The work deals with the clinical analysis of the richest experience in the surgical treatment of patients with generalized myasthenia in the country. The efficacy of various methods for the diagnosis of affection of the thymus is studied comparatively. It is shown that for more effective diagnosis of neoplastic lesions of the thymus wider use of computerized and magneto-resonance tomography is expedient and that they must replace the insufficiently informative and invasive examination methods used today (pneumonodiastinography, phlebography, scintigraphy, etc.) which still form the basis for the system of diagnostic research in patients with myasthenia. The authors determined the indications and contraindications for, the optimum terms for undertaking the surgical intervention and its volume in neoplastic and nonneoplastic diseases of the thymus in adults and children. Comparative evaluation of various means of preoperative management was conducted. It is shown that to improve the patient's condition and stabilize the myasthenic status on the possibly minimal doses of anticholesteremic agents glucocorticoid hormones should be included in the complex of preoperative management. The indications for hormonotherapy were determined. The efficacy of plasmapheresis, radiotherapy, and glomectomy with denervation of the sinocarotid zone was evaluated and their place in the complex treatment of patients was determined. From study of the late-term results of treatment of a large group of patients (children, adults with a thymus) the main causes of unsuccessful surgical treatment of generalized myasthenia were identified and the concrete means of improving the results of treatment were planned. Two types of changes in the clinico-immuno-morphological values were revealed in patients with generalized myasthenia, differing evidently in pathogenesis. New clinico-immuno-morphological correlations and prognostic factors were discovered, and a method for prognosticating the effect of thymectomy was suggested. It is shown that splenectomy has a favorable effect in the most severe category of patients in unsuccessful operative and nonoperative treatment. The tactics of management of patients accepted in the clinic led to a uncomplicated course of the postoperative period in the majority of patients in the last years.

Adult↗
PubMed1992

Interaction of lambda cro repressor with synthetic operator OR3 studied by competition binding with minor groove binders.

In the present work, we employ a combination of CD spectroscopy and gel retardation technique to characterize thermodynamically the binding of lambda phage cro repressor to a 17 base pair operator OR3. We have found that three minor groove-binding antibiotics, distamycin A, netropsin and sibiromycin, compete effectively with the cro for binding to the operator OR3. Among these antibiotics, sibiromycin binds covalently to DNA in the minor groove at the NH2 of guanine, whereas distamycin A and netropsin interact preferentially with runs of AT base pairs and avoid DNA regions containing guanine bases in the two polynucleotide strands. Only subtle DNA conformation changes are known to take place upon binding of these antibiotics. Both the CD spectral profiles and the results of the gel retardation experiments indicate that distamycin A and netropsin can displace cro repressor from the operator OR3. The binding of cro repressor to the OR3 is accompanied by considerable changes in CD in the far-UV region which appear to be attributed to a DNA-dependent structural transition in the protein. Spectral changes are also induced in the wavelength region of 270-290 nm. The CD spectral profile of the cro-OR3 mixture in the presence of distamycin A can be represented as a sum of the CD spectrum of the repressor-operator complex and spectrum of distamycin-DNA complex at the appropriate molar ratio of the bound antibiotic to the operator DNA (r). When r tends to the saturation level of binding the CD spectrum in the region of 270-360 nm approaches a CD pattern typical of complexes of the antibiotic with the free DNA oligomer. This suggests that simultaneous binding of cro repressor and distamycin A to the same DNA oligomer is not possible and that distamycin A and netropsin can be used to determine the equilibrium affinity constant of cro repressor to the synthetic operator from competition-type experiments. The binding constant of cro repressor to the OR3 is found to be (6 +/- 1).10(6)M-1 at 20 degrees C in 10 mM sodium cacodylate buffer (pH 7.0) in the presence of 0.1 M NH4F.

Aminoglycosides↗
PubMed1991

Two-stage thermal unfolding of [Cys55]-substituted Cro repressor of bacteriophage lambda.

It has been shown by scanning calorimetry and 1H NMR spectroscopy that thermal denaturation of mutant lambda phage cro repressor in which Val55 was substituted for Cys, proceeds in 2 stages in contrast to the wild type protein. At neutral pH values, an additional cooperative transition has been observed at about 100 degrees C. Calorimetric measurements on the mutant and its tryptic fragment lead to the conclusion that the two-stage character of thermal unfolding of the mutant is due to a disruption of an additional cooperative domain in the dimer molecule which is stabilized by the S-S crosslink.

Bacteriophage lambda↗
PubMed1991

[Intraoperative ultrasonographic examination in surgery of the biliary tract].

The diagnostic value of intraoperative ultrasonic examination (IUSE) in surgery on the biliary tract was shown from analysis of the literature and the authors' experience. IUSE in more than 300 patients demonstrated a high resolving power in the diagnosis of choledocholithiasis and the absence of radiation load, allergic reactions and complications. In comparison of IUSE with intraoperative cholangiography in the diagnosis of stones in the gallbladder the total efficacy of the methods was 98.2% and 92.6%, respectively. IUSE has no side effects, is a simpler and cheaper method of examination, and allows goal-oriented removal of stones from the biliary tract. It is shown that with accumulation of experience in IUSE the performance of intraoperative cholangiography can be brought to the minimum.

Adult↗
PubMed1991

[Diagnosis and surgical treatment of myasthenia gravis in childhood].

Thirty-year experience in surgical treatment of myasthenia in children and adolescents (175 patients) is generalized. It provides evidence of delayed recognition of this disease in many patients and of prolonged, often ineffective nonoperative treatment. Operation for thymectomy should be one of the main methods for the management of myasthenia in children. Operative treatment is indicated in a disease of moderate severity or in a severe course of myasthenia, particularly in patients with the first type of immunomorphological changes. It is important to amend the myasthenic disorders before the operation by means of maximally possible doses of anticholinesterases with addition of corticosteroids, whenever necessary. Whatever the method of treatment chosen, the patients must be kept under skilled dynamic control. Excellent and good late-term results were produced in 84.6% of cases.

Adolescent↗
PubMed1984

Interactions between cro repressor and the model specific binding site.

Binding of lambda phage cro repressor to the synthetic half of OR3, the most conservative half of the specific binding sites, was investigated by proton nuclear magnetic resonance spectroscopy. It was found that the alpha-helical segment (27-36) of the protein was involved in specific interactions with the model binding site. The 3-dimensional structure of cro repressor does not change noticeably upon complex formation. Intercalation can be excluded as a possible means of interaction.

Bacteriophage lambda↗
PubMed1984

1H NMR study of the interaction of bacteriophage lambda Cro protein with the OR3 operator. Evidence for a change of the conformation of the OR3 operator on binding.

The specific complex between the lambda phage OR3 operator and the Cro protein has been studied by proton NMR spectroscopy at 500 MHz. The DNA imino proton resonances of this complex have been assigned to specific base pairs using the known assignments of these resonances for the free operator. Increase of the protein/DNA ratio to complete saturation of the OR3 operator with the Cro protein made it possible to follow the shift changes of the resonances. Ambiguities were resolved by nuclear Overhauser effect measurements on the complex. The shifts of the imino proton resonance positions provide information on the changes induced in the conformation of the operator upon complex formation with a dimer of the Cro protein. The most striking shift occurs for the central (GC 9) base pair, which is known to have no direct contacts with the Cro protein. This shift may be induced by a bend in the OR3 operator DNA at the GC 9 base pair to accommodate the operator for the binding of the Cro protein dimer. The imino proton resonances of two additional base pairs can be observed in the complex, demonstrating an overall stabilization of the DNA structure by the binding of the Cro protein.

Bacteriophage lambda↗