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Biomedical subjects

G Otting

Publications and source records attributed to G Otting.

At least 37 records · Page 2Linked to original sources
PubMed1999

Solvation study of the non-specific lipid transfer protein from wheat by intermolecular NOEs with water and small organic molecules.

Intermolecular nuclear Overhauser effects (NOEs) were measured between the protons of various small solvent or gas molecules and the non-specific lipid transfer protein (ns-LTP) from wheat. Intermolecular NOEs were observed with the hydrophobic pocket in the interior of wheat ns-LTP, which grew in intensity in the order cyclopropane (saturated solution) < methane (140 bar) < ethane (40 bar) < acetonitrile (5% in water) < cyclohexane (saturated solution) < benzene (saturated solution). No intermolecular, NOEs were observed with dioxane (5% in water). The intermolecular NOEs were negative for all of the organic molecules tested. Intermolecular NOEs between wheat ns-LTP and water were weak or could not be distinguished from exchange-relayed NOEs. As illustrated by the NOEs with cyclohexane versus dioxane, the hydrophobic pocket in wheat ns-LTP preferably binds non-polar molecules. Yet, polar molecules like acetonitrile can also be accommodated. The pressure dependence of the NOEs between methane and wheat ns-LTP indicated incomplete occupancy, even at 190 bar methane pressure. In general, NOE intensities increased with the size of the ligand molecule and its vapor pressure. NMR of the vapor phase showed excellent resolution between the signals from the gas phase and those from the liquid phase. The vapor concentration of cyclohexane was fivefold higher than that of the dioxane solution, supporting the binding of cyclohexane versus uptake of dioxane.

Acetonitriles↗
PubMed1998

Random coil conformation of a Gly/Ala-rich insert in IkappaB alpha excludes structural stabilization as the mechanism for protection against proteasomal degradation.

Peptide segments of multiple glycine and alanine residues prevent the proteolytic degradation of ubiquitinated proteins by the proteasome. The structure of a Gly/Ala-rich insert in IkappaB alpha was probed by nuclear magnetic resonance (NMR) spectroscopy, comparing IkappaB alpha samples with and without Gly/Ala-rich insert. Narrow 1H-NMR resonances at chemical shifts indicative of random coil conformations were observed in the difference spectrum. circular dichroism (CD) measurements further confirm that the mechanism of protection against proteolytic degradation is not based on structural transition or stabilization caused by the Gly/Ala-rich segment. In addition, most of the N- and C-terminal residues outside the ankyrin repeats in wild-type IkappaB alpha were found to be flexibly disordered.

Alanine↗
PubMed1998

Water molecules in DNA recognition I: hydration lifetimes of trp operator DNA in solution measured by NMR spectroscopy.

The present NMR study investigates the residence times of the hydration water molecules associated with uncomplexed trp operator DNA in solution by measuring intermolecular nuclear Overhauser effects (NOE) between water and DNA protons, and the nuclear magnetic relaxation dispersion (NMRD) of the water 2H and 17O resonances. Both methods indicate that the hydration water molecules exchange with bulk water on the sub-nanosecond time scale at 4 degreesC. No evidence was obtained for water molecules bound with longer residence times. In particular, the water molecules at the sites of interfacial hydration in the trp repressor/operator complex do not seem kinetically stabilized in the uncomplexed DNA. Analysis of the crystal structures of two different trp repressor/operator complexes shows very similar structural environments for the water molecules mediating specific contacts between the protein and the DNA, whereas much larger variations are observed for the location of corresponding water molecules detected in the crystal structure of an uncomplexed trp operator DNA duplex. Therefore, it appears unlikely that the hydration characteristics of the uncomplexed DNA target would be a major determinant of trp repressor/operator recognition.

Bacterial Proteins↗
PubMed1998

Water molecules in DNA recognition II: a molecular dynamics view of the structure and hydration of the trp operator.

The structure and hydration of the DNA duplex d-(AGCGTACTAGTACGCT)2 corresponding to the trp operator fragment used in the crystal structure of the half site complex (PDB entry 1TRR) was studied by a 1.4 ns molecular dynamics simulation in water. The simulation, starting from a B-DNA conformation, used a non-bonded cutoff of 1.4 nm with a reaction field correction and resulted in a stable trajectory. The average DNA conformation obtained was closer to the ones found in the crystal structures of the complexes (PDB entries 1TRO and 1TRR) than to the crystal structure of unbound trp operator (Nucleic Acid Database entry BDJ061). The DNA hydration was characterized in terms of hydrogen bond percentages and corresponding residence times. The residence times of water molecules within 0.35 nm of the DNA non-exchangeable protons were calculated for comparison with NMR measurements of intermolecular water-DNA NOEs and nuclear magnetic relaxation dispersion measurements. No significant difference was found between major and minor groove hydration. The DNA donors and acceptors were hydrogen bonded to water molecules for 77(+/-19)% of the time on average. The average residence time of the hydrogen bonded water molecules was 11(+/-11) ps with a maximum of 223 ps. When all water molecules within NOE distance (0.35 nm) of non-exchangeable protons were considered, the average residence times increased to an average of 100(+/-4) ps and a maximum of 608 ps. These results agree with the experimental NMR results of Sunnerhagen et al. which did not show any evidence for water molecules bound with more than 1 ns residence time on the DNA surface. The exchange of hydration water from the DNA occurred in the major groove primarily through direct exchange with the bulk solvent, while access to and from the minor groove frequently proceeded via pathways involving ribose O3' and O4' and phosphate O2P oxygen atoms. The most common water diffusion pathways in the minor groove were perpendicular to the groove direction. In general, water molecules visited only a limited number of sites in the DNA grooves before exiting. The hydrogen bonding sites, where hydrogen bonds could be formed with donor and acceptor groups of the DNA, were filled with water molecules with an average B-factor value of 0.58 mn2. No special values were observed at any of the sites, where water molecules were observed both in the trp repressor/operator co-crystals and in the crystal structure of unbound DNA.

Bacterial Proteins↗
PubMed1998

Lipid membrane binding of NK-lysin.

The membrane-binding properties and pore-forming potential of the tumor-lysing and antibacterial polypeptide NK-lysin were investigated. Fluorescence quenching experiments show a drastic change of accessibility to Trp58 in solution and in association with a lipid membrane. Calcein release from large unilamellar vesicles and fluctuating conductivity observed across a planar lipid bilayer of asolectin show that NK-lysin renders lipid bilayers permeable in a transient fashion, indicating a nonspecific lipid interaction as the mechanism underlying the biological activity. FTIR experiments show the same amount and type of regular secondary structure of NK-lysin in the membrane as in aqueous solution and exclude a structural rearrangement into a set of parallel or antiparallel alpha-helices as the predominant conformation. The molecular mechanism of the membrane-destabilizing effect of NK-lysin is discussed.

Anti-Infective Agents↗
PubMed1998

An alpha/beta-HSQC-alpha/beta experiment for spin-state selective editing of IS cross peaks

A generalized version of the TROSY experiment allows the spin-state selective editing of the four multiplet components of 15N-1H cross peaks of amide groups in proteins into four different subspectra, with no penalty in sensitivity. An improvement by 2 in sensitivity results, if only two of the four multiplet components are selected. Use of the experiment for the measurement of 1JHN coupling constants is discussed. A water flip-back version of the experiment is demonstrated with a 45 kDa fragment of 15N/2H labeled Staphylococcus aureus gyrase B. Copyright 1998 Academic Press.

Journal Article↗
PubMed1998

Isolation and structure of a new galactolipid from oat seeds.

Seeds of oat (Avena sativa L.) were recently shown to contain significant quantities of a new hydroxy acid, (15 R)-hydroxy-(9Z),(12Z)-octadecadienoic acid (trivial name, avenoleic acid). In the present work, avenoleate was found to be mainly (63%) localized in the glycolipid fraction of oat seed lipids. Fractionation of the glycolipids by thin-layer chromatography and reversed-phase high-performance liquid chromatography revealed the presence of a main molecular species which accounted for 20% of the total avenoleate content of oat seeds. Structural studies by chemical methods and mass spectrometry demonstrated that the avenoleate-containing glycolipid was a galactolipid assembled of one molecule of avenoleic acid, two molecules of linoleic acid, two molecules of D-galactose, and one molecule of glycerol. Degradation of the new galactolipid by chemical and enzymatic methods demonstrated the localization of acyl chains, i.e., linoleate at sn-1 and linoleoylavenoleate at sn-2. Nuclear magnetic resonance spectroscopy gave independent support for this structure and also demonstrated that the two galactoses formed an alpha-D-galactopyranosyl-1-6-beta-D-galactopyranosyl moiety which was bound to the sn-3 position. Based on these experiments, the new galactolipid could be formulated as 1-[(9'Z),(12'Z)-octadecadienoyl]-2-[(15''R)-[(9'''Z),(12'''Z)-o ctadecadienoyloxy]-(9''Z),(12''Z)-octadecadienoyl]-3-(alpha-D-g alactopyranosyl-1-6-beta-D-galactopyranosyl)-glycerol. Quantitatively, the amount of the avenoleate-containing galactolipid was of the same order of magnitude as those of individual molecular species of digalactosyldiacylglycerol containing nonoxygenated acyl chains. The content of the new galactolipid in oat seeds was 0.5-0.6 mg per g of seed.

Avena↗
PubMed1998

Heteronuclear correlation experiments for the determination of one-bond coupling constants.

Spin-state selective experiments, HSQC-alpha/beta and CT-HMQC-alpha/beta, are proposed for the simple and rapid measurement of scalar one-bond coupling constants in two-dimensional, 1H-detected 15N-1H or 13C-1H correlation experiments based on HSQC and HMQC schemes. Pairs of subspectra are obtained, containing either the high-field or the low-field component of the doublet representing the one-bond coupling constant. The subspectral editing procedure retains the full sensitivity of HSQC and HMQC spectra recorded without heteronuclear decoupling during data acquisition, with a spectral resolution similar to that of decoupled spectra.

Bacterial Proteins↗
PubMed1998

HMQC and HSQC experiments with water flip-back optimized for large proteins.

An HMQC experiment is proposed, dubbed FHMQC, where water flip-back is achieved by a single water-selective pulse preceding the basic HMQC pulse sequence. The scheme is demonstrated with a 15N,1H-HMQC spectrum of uniformly 15N/2H-labelled S. aureus DNA gyrase B with a molecular weight of 45 kDa for the unlabelled protein. The sensitivity of the experiment is improved compared to that of an FHSQC spectrum. It is further shown that the original FHSQC experiment can be shortened by the use of bipolar gradients. Relaxation times of different 15N magnetizations and coherences were measured. The new FHMQC scheme is implemented in 3D NOESY-15N-HMQC and 3D 15N-HMQC-NOESY-15N-HMQC pulse sequences which are demonstrated with a 24 kDa fragment of uniformly 15N/13C/2H-labelled S. aureus DNA gyrase B.

Bacterial Proteins↗
PubMed1998

Spin-state selection filters for the measurement of heteronuclear one-bond coupling constants.

Novel alpha/beta-half-filter elements are proposed for the separation of the high-field and low-field component of 1JHC and 1JHN splittings into different subspectra. The alpha/beta-half-filter elements are of the same duration as the S3CT pulse sequence element and, like this, are less sensitive to cross talk between different subspectra than the original shorter alpha/beta-half-filters. The filter elements are demonstrated with the measurement of 1JHC coupling constants of C alpha H groups in 2D and 3D experiments and the subspectral editing of the four different multiplet components observed in two-dimensional alpha/beta-HSQC-alpha/beta spectra recorded without heteronuclear decoupling in either dimension.

Carbon Isotopes↗
PubMed1998

Rapid measurement of scalar three-bond 1HN-1H alpha spin coupling constants in 15N-labelled proteins.

Two new 2D NMR experiments, CT-HMQC-HA and CT-HMQC-HN, are proposed for the rapid measurement of homonuclear 3JHNH alpha coupling constants of uniformly 15N-enriched proteins in solution. The experiments are based on the comparison of the signal intensities in a pair of constant-time [15N,1H]-HMQC spectra recorded with and without decoupling of the amide proton-alpha proton coupling. Experimental data recorded with the 78-residue N-terminal domain of the E. coli arginine repressor (ArgR-N) and with oxidized E. coli flavodoxin (176 residues) showed good agreement with 3JHNH alpha coupling constants obtained by fitting of the multiplet fine structure of the amide proton resonances or from a 3D HNHA-J experiment, respectively. Quantitative estimates for the effects from different relaxation rates of in-phase and antiphase magnetization are given.

Amides↗
PubMed1997

NMR structure of the death domain of the p75 neurotrophin receptor.

The intracellular domain of the p75 neurotrophin receptor (p75ICD) lacks catalytic activity but contains a motif similar to death domains found in the cytoplasmic regions of members of the tumor necrosis factor receptor family and their downstream targets. Although some aspects of the signaling pathways downstream of p75 have been elucidated recently, mechanisms of receptor activation and proximal signaling events are unknown. Here we report the nuclear magnetic resonance (NMR) structure of the 145 residue long p75ICD. The death domain of p75ICD consists of two perpendicular sets of three helices packed into a globular structure. The polypeptide segment connecting the transmembrane and death domains as well as the serine/threonine-rich C-terminal end are highly flexible in p75ICD. Unlike the death domains involved in signaling by the TNF receptor and Fas, p75ICD does not self-associate in solution. A surface area devoid of charged residues in the p75ICD death domain may indicate a potential site of interaction with downstream targets.

Amino Acid Sequence↗
PubMed1997

NMR assignments, secondary structure and hydration of oxidized Escherichia coli flavodoxin.

Recombinant flavodoxin from Escherichia coli was uniformly enriched with 15N and 13C isotopes and its oxidized form in aqueous solution investigated by three-dimensional NMR spectroscopy. Nearly complete 1H, 15N and 13C resonance assignments were obtained. The secondary structure was determined from chemical shift, NOE and 3J(HNH alpha) coupling constant data. Like homologous long-chain flavodoxins, E. coli flavodoxin contains a five-stranded parallel beta-sheet and five helices. The beta-strands were found to comprise the residues 3-8, 29-34, 48-56, 80-89, 114-116 and 141-145. The helices comprise residues 12-25, 40-45, 62-73, 98-108 and 152-166. The FMN-binding site was determined by intermolecular NOEs and low-field shifted amide proton resonances induced by the phosphoester group of the cofactor. The data are in good agreement with a previously predicted model of E. coli flavodoxin [Havel, T. F. (1993) Mol. Sim. 10, 175-210]. The analysis of of water-flavodoxin NOEs revealed the presence of two, possibly three, buried hydration water molecules which are located at sites, where homologous flavodoxins from Anacystis nidulans and Anabena 7120 contain conserved hydration water molecules. One of these water molecules mediates hydrogen bonds between the protein backbone and the ribityl chain of the FMN cofactor.

Amino Acid Sequence↗
PubMed1997

Precise limits of the N-terminal domain of DnaB helicase determined by NMR spectroscopy.

Two separate N-terminal fragments of the 470-amino-acid Escherichia coli DnaB helicase, comprising residues 1-142 and 1-161, were expressed in E. coli. The proteins were extracted in a soluble fraction, purified, and characterised physically. In contrast to the full-length protein, which is hexameric, both fragments exist as monomers in solution, as demonstrated by sedimentation equilibrium measurements. CD spectroscopy was used to confirm that the 161-residue fragment is highly structured (mostly alpha-helical) and undergoes reversible thermal denaturation. The structurally well-defined core of the N-terminal domain of the DnaB helicase is composed of residues 24 to 136, as determined by assignment of resonances from flexible residues in NMR spectra. The 1H NMR signals of the flexible residues are located at random coil chemical shifts, and their linewidths are significantly narrower than those of the structured core, indicating complete disorder and increased mobility on the nanosecond time scale. The results support the idea of a flexible hinge region between the N- and C-terminal domains of the native hexameric DnaB protein.

Bacterial Proteins↗
PubMed1997

Organic solvents identify specific ligand binding sites on protein surfaces.

Enzymes frequently recognize substrates and pharmaceutical drugs through specific binding interactions in deep pockets on the protein surface. We show how the specificity-determining substrate binding site of hen egg-white lysozyme (HEWL) can be readily identified in aqueous solution by nuclear magnetic resonance spectroscopy using small organic solvent molecules as detection probes. Exchange of magnetization between the 1H nuclei of the protein and the ligands through dipole-dipole interactions is observed which allows the modeling of their position and orientation at the binding site. Combined with site-specific binding constants measured by titration experiments with different organic solvents, the method can provide important information for rational drug design. In addition, the lifetime of nonspecific interactions of HEWL with organic solvents is shown to be in the sub-nanosecond time range.

Animals↗
PubMed1997

NMR identification of hydrophobic cavities with low water occupancies in protein structures using small gas molecules.

Magnetization transfer through dipole-dipole interactions (nuclear Overhauser effects, NOEs) between water protons and the protons lining two small hydrophobic cavities in hen egg-white lysozyme demonstrates the presence of water molecules with occupancies of approximately 10-50%. Similarly, NOEs were observed between the cavity protons and the protons of hydrogen, methane, ethylene or cyclopropane applied at 1-200 bar pressure. These gases can thus be used as general NMR indicators of empty or partially hydrated hydrophobic cavities in proteins. All gases reside in the cavities for longer than 1 ns in marked contrast to common belief that gas diffusion in proteins is not much slower than in water. Binding to otherwise empty cavities may be a major aspect of the anesthetic effect of small organic gas molecules.

Algorithms↗