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D Uhrín

Publications and source records attributed to D Uhrín.

At least 19 recordsLinked to original sources
PubMed2000

Location of cyanine-3 on double-stranded DNA: importance for fluorescence resonance energy transfer studies.

Fluorescence resonance energy transfer provides valuable long-range distance information about macromolecules in solution. Fluorescein and Cy3 are an important donor-acceptor pair of fluorophores; the characteristic Förster length for this pair on DNA is 56 A, so the pair can be used to study relatively long distances. Measurement of FRET efficiency for a series of DNA duplexes terminally labeled with fluorescein and Cy3 suggests that the Cy3 is close to the helical axis of the DNA. An NMR analysis of a self-complementary DNA duplex 5'-labeled with Cy3 shows that the fluorophore is stacked onto the end of the helix, in a manner similar to that of an additional base pair. This provides a known point from which distances calculated from FRET measurements are measured. Using the FRET efficiencies for the series of DNA duplexes as restraints, we have determined an effective position for the fluorescein, which is maximally extended laterally from the helix. The knowledge of the fluorophore positions can now be used for more precise interpretation of FRET data from nucleic acids.

Base Sequence↗
PubMed2000

Structural changes accompanying pH-induced dissociation of the beta-lactoglobulin dimer.

We have used NMR spectroscopy to determine the three-dimensional (3D) structure, and to characterize the backbone dynamics, of a recombinant version of bovine beta-lactoglobulin (variant A) at pH 2. 6, where the protein is a monomer. The structure of this low-pH form of beta-lactoglobulin is very similar to that of a subunit within the dimer at pH 6.2. The root-mean-square deviation from the pH 6.2 (crystal) structure, calculated for backbone atoms of residues 6-160, is approximately 1.3 A. Differences arise from the orientation, with respect to the calyx, of the A-B and C-D loops, and of the flanking three-turn alpha-helix. The hydrophobic cavity within the calyx is retained at low pH. The E-F loop (residues 85-90), which moves to occlude the opening of the cavity over the pH range 7.2-6.2, is in the "closed" position at pH 2.6, and the side chain of Glu89 is buried. We also carried out measurements of (15)N T(1)s and T(2)s and (1)H-(15)N heteronuclear NOEs at pH 2.6 and 37 degrees C. Although the residues of the E-F loop (residues 86-89) have the highest crystallographic B-factors, the conformation of this loop is reasonably well defined by the NMR data, and its backbone is not especially mobile on the pico- to nanosecond time scale. Several residues (Ser21, Lys60, Ala67, Leu87, and Glu112) exhibit large ratios of T(1) to T(2), consistent with conformational exchange on a micro- to millisecond time scale. The positions of these residues in the 3D structure of beta-lactoglobulin are consistent with a role in modulating access to the hydrophobic cavity.

Animals↗
PubMed2000

3D HCCH(3)-TOCSY for resonance assignment of methyl-containing side chains in (13)C-labeled proteins.

Two 3D experiments, (H)CCH(3)-TOCSY and H(C)CH(3)-TOCSY, are proposed for resonance assignment of methyl-containing amino acid side chains. After the initial proton-carbon INEPT step, during which either carbon or proton chemical shift labeling is achieved (t(1)), the magnetization is spread along the amino acid side chains by a carbon spin lock. The chemical shifts of methyl carbons are labeled (t(2)) during the following constant time interval. Finally the magnetization is transferred, in a reversed INEPT step, to methyl protons for detection (t(3)). The proposed experiments are characterized by high digital resolution in the methyl carbon dimension (t(2max) = 28.6 ms), optimum sensitivity due to the use of proton decoupling during the long constant time interval, and an optional removal of CH(2), or CH(2) and CH, resonances from the F(2)F(3) planes. The building blocks used in these experiments can be implemented in a range of heteronuclear experiments focusing on methyl resonances in proteins. The techniques are illustrated using a (15)N, (13)C-labeled E93D mutant of Schizosacharomyces pombe phosphoglycerate mutase (23.7 kDa).

Magnetic Resonance Spectroscopy↗
PubMed1998

Sensitivity- and gradient-enhanced hetero (omega1) half-filtered TOCSY experiment for measuring long-range heteronuclear coupling constants.

An enhanced version of the X (omega1) half-filtered TOCSY experiment for measurement of long-range heteronuclear coupling constants is proposed which yields high-quality spectra with substantially increased sensitivity and resolution. The modified method features gradient-enhanced X filtering sequences, broadband homonuclear decoupling during t1, optional 1JXH scaling in the F1 domain, and gradient coherence selection in combination with the sensitivity-enhanced protocol for the TOCSY transfer. These modifications extend the applicability of the method--coupling constants can be measured accurately for natural abundance samples at low concentrations and for compounds yielding complex spectra. Computer-aided analysis of E.COSY-type multiplets is applied for the determination of heteronuclear long-range coupling constants.

Magnetic Resonance Spectroscopy↗
PubMed1998

Gradient- and sensitivity-enhanced TOCSY experiments.

A pulsed field gradient version of the sensitivity-enhanced 2D TOCSY experiment is proposed which yields high-quality spectra with improved sensitivity and a minimum of two scans per t1 increment. For rapid acquisition of 1D TOCSY spectra, the 1D DPFGSE-TOCSY experiment was modified to include phase-encoded multiple-selective excitation followed by a simple spectral editing. Combination of these two building blocks is used in a sensitivity-enhanced 2D analog of the 3D TOCSY-TOCSY experiment which provides an efficient tool for resolving severely overlapped signals of oligomers in short experimental time.

Analgesics, Opioid↗
PubMed1998

Complete assignment of 1H, 13C and 15N chemical shifts for bovine beta-lactoglobulin: secondary structure and topology of the native state is retained in a partially unfolded form.

Although beta-lactoglobulin (beta-LG) has been studied extensively for more than 50 years, its physical properties in solution are not yet understood fully in terms of its three-dimensional (3D) structure. For example, despite a recent high-resolution crystal structure, it is still not clear why the two common variants of bovine beta-LG which differ by just two residues have different aggregation properties during milk processing. We have conducted solution-state NMR studies on a recombinant form of the A variant of beta-LG at low pH conditions where the protein is partially unfolded and exists as a monomer rather than a dimer. Using a 13C, 15N-labelled sample, expressed in Pichia pastoris, we have employed the standard combination of 3D heteronuclear NMR techniques to obtain near complete assignments of proton, carbon and nitrogen resonances. Using a novel pulse sequence we were able to obtain additional assignments, in particular those of methyl groups in residues preceding proline within the sequence. From chemical shifts and on the basis of inter-residue NOEs, we have inferred the secondary structure and topology of monomeric beta-LG A. It includes eight antiparallel beta-strands arranged in a barrel, flanked by an alpha-helix, which is typical of a member of the lipocalin family. A detailed comparison with the crystal structure of the dimeric form (for a mixture of A and B variants) at pH 6.5 reveals a close resemblance in both secondary structure and overall topology. Both forms have a ninth beta-strand which, at the higher pH, forms part of the dimer interface. These studies represent the first full NMR assignment of beta-LG and will form the basis for a complete characterisation of the solution structure and dynamics of this protein and its variants.

Amino Acid Sequence↗
PubMed1997

Structural basis of the Neisseria meningitidis immunotypes including the L4 and L7 immunotypes.

The application of high-resolution 1H, 13C and 31P NMR and MS analyses to the oligosaccharide moieties of the L4 and L7 immunotypes of Neisseria meningitidis revealed that they had the following structures: [formula: see text] The fact that the L7 LPS is not sialylated at O-3 of its terminal beta-D-galactopyranosyl residue implies that it is a mutant strain unable to endogenously sialylate its lacto-N-neotetraose antenna. With the structural elucidation of the L4 and L7 LPS immunotypes, a more comprehensive structural profile of the LPS involved in disease isolates can now be assembled. This provides valuable insights into the structural basis of the N. meningitidis immunotyping system which could be of use in formulating an LPS-based vaccine against meningococcal meningitis.

Antigens, Bacterial↗
PubMed1997

Sensitivity- and gradient-enhanced heteronuclear coupled/decoupled HSQC-TOCSY experiments for measuring long-range heteronuclear coupling constants.

A pulsed field gradient version of the sensitivity-enhanced 2D HSQC-TOCSY experiment is proposed for measurement of long-range heteronuclear coupling constants. The coupling constants are obtained by computer-aided analysis of mixed-phase multiplets with and without the heteronuclear splitting. Generation of pure phase data is not required. Since large 1JXH and JHH couplings are used for coherence transfer, small nJXH can be measured accurately, which could be difficult to obtain from purely heteronuclear polarization transfer experiments.

Data Interpretation, Statistical↗
PubMed1996

Structural and immunochemical characterization of the type VIII group B Streptococcus capsular polysaccharide.

The type VIII capsular polysaccharide has been isolated and purified from a newly described strain of group B Streptococcus which is a leading cause of sepsis and neonatal meningitis in Japan. The polysaccharide contains D-glucose, D-galactose, L-rhamnose, and sialic acid in the molar ratio 1:1:1:1. By means of high resolution 1H nuclear magnetic resonance (1H NMR), 13C NMR, and homo- and heterocorrelated NMR, the repeating unit structure of the type VIII polysaccharide was delineated as the following, [formula: see text] Enzymatic studies established this polysaccharide as the first from which sialic acid, linked to a branched beta-D-galactopyranosyl residue, is known to be removed by bacterial neuraminidase.

Antigens, Bacterial↗
PubMed1995

Structural elucidation of the capsular polysaccharide of Bacteroides fragilis strain 23745M1.

The capsule of Bacteroides fragilis (ATCC23745) consists of two distinct polysaccharides, the separation of which could not be accomplished. The mouse-passaged strain (23745M1), however, yielded a preponderant polysaccharide which was isolated and purified. Using mainly high resolution NMR spectroscopy, the structure of the polysaccharide was elucidated and it is composed of the following repeating unit: [formula see text]

Acetylglucosamine↗
PubMed1994

Application of 1D and 2D NMR techniques to the structure elucidation of the O-polysaccharide from Proteus mirabilis O:57.

The LPS O-polysaccharide (O-PS) produced by Proteus mirabilis serotype O:57 (ATCC 49995) was shown by NMR spectroscopy and chemical analysis to be a high-molecular-weight acidic branched polymer of pentasaccharide repeating units, composed of D-glucose, D-galactose, 2-acetamido-2-deoxy-D-galactose and glycerophosphate residues (1:2:2:2:1). Application of one- and two-dimensional NMR methods allowed the complete assignment of notoriously crowded 1H and 13C spectra of the O-PS, leading to the determination of its structure. Several of the NMR techniques used were applied for the first time to the structure elucidation of polysaccharides. The resonances of galactose H5, H6 and H6' were identified by a 1D analog of 3D NOESY-TOCSY and 2D (1H,1H) triple-quantum experiments. The position and the nature of the phosphate group were determined from 2D 31P (omega 1)-half-filtered COSY and 2D 31P-relayed COSY spectra. 2D HMQC-TOCSY and 2D single-quantum proton-carbon long-range correlation techniques were used to overcome the difficulties of severe overlap and higher order effects in the 1H NMR spectrum of the O-PS. The latter technique, together with 2D NOESY, enabled us to identify the substitution positions, the anomeric configurations and the sequence of the component glycose residues in the O-PS.

Carbohydrate Conformation↗
PubMed1993

Structure of the sialylated L3 lipopolysaccharide of Neisseria meningitidis.

The L3 immunotype lipopolysaccharide (LPS) of Neisseria meningitidis was subjected to degradation procedures, which produced a number of different oligosaccharide fragments. The high resolution 1H and 13C NMR spectroscopic analyses of these oligosaccharides yielded structural information on a number of different regions of the LPS. For example, from one oligosaccharide, it was found that the endogenous sialylation of the meningococcal LPS occurs at O-3 of the terminal beta-D-galactopyranosyl residue of its lacto-N-neotetraose antenna in the alpha-D-configuration. From another, it was also established that the dominant structural feature responsible for L3 epitope specificity is the presence of a phosphorylethanolamine substituent at O-3 of the penultimate heptopyranosyl residue of its other antenna. In addition from information obtained with another oligosaccharide the structure of the 3-deoxy-D-manno-octulosonic acid disaccharide region of the L3 LPS was also elucidated. From all the above cumulative data plus some published data, it was then possible to reconstruct the complete structure of the entire native L3 LPS.

Carbohydrate Conformation↗
PubMed1993

Vermiculin derivatives. Part 1: Synthesis of vermiculin derivatives.

Hydrogenation of the macrodiolide antibiotic vermiculin (1) over Adams catalyst afforded [8S, 16S]-8,16-bis(2'-oxopropyl)-1,9- dioxyacyclohexadeca-2,5,10,13-tetrone (2), [8S, 16S]-8,16-bis(2'-oxopropyl)-13-hydroxy-1,9-dioxacyclohexadeca- 2,5,10-trione (3), [8S,16S]-8,16-bis(2' oxopropyl)-1,9- dioxacyclohexadeca-2,5,10-trione (4) together with [7S]-4,9-dioxo-7-(4',9'-dioxodecanoyloxy)decanoic acid (5). Hydrogenation of diolide 1 over Pd/C gave tetrahydrovermiculin (2) only. The prepared compounds showed lower antibacterial and cytotoxic activities than vermiculin (1).

Animals↗
PubMed1992

Vermixocins A and B, two novel metabolites from Penicillium vermiculatum.

Vermixocins A and B, 3-(1'-hydroxy-3'-methylbutyl)- and 3-(1'-acetoxy-3'-methylbutyl)-11-hydroxy-4-methoxy-9-methyl-5H,7H- dibenzo[c,f][1,5]dioxocin-5-one, respectively, were isolated from the mycelium of Penicillium vermiculatum. Both metabolites showed cytotoxic effects on lympholeukemia cells P388.

Animals↗
PubMed1990

Regulation of biosynthesis of thiolutin and aureothricin in Streptomyces kasugaensis.

L-Methionine and DL-ethionine decreased production of thiolutin and aureothricin in Streptomyces kasugaensis. In the presence of L-methionine the culture also produced 3-methylthioacrylic acid, 3-methylthiopropionic acid and 3,6-bis-(2-methylthioethyl)-2,5-dioxopiperazine. Production of the metabolites depended on the concentration of L-methionine in the medium.

Anti-Bacterial Agents↗