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Biomedical subjects

G Otting

Publications and source records attributed to G Otting.

At least 19 recordsLinked to original sources
PubMed2001

Hydrophobic interactions in a cyanobacterial plastocyanin-cytochrome f complex.

The complex of the photosynthetic redox partners plastocyanin and cytochrome f from the thermophilic cyanobacterium, Phormidium laminosum, was investigated by nuclear magnetic resonance (NMR). Chemical-shift perturbation analysis of amide proton and nitrogen nuclei implicates the hydrophobic patch and, to a lesser extent, the "eastern face" of plastocyanin in the complex interface. Intermolecular pseudocontact shifts observed in the complex of cadmium-substituted plastocyanin and ferric cytochrome f specifically define the site of interaction to be between the hydrophobic patch of plastocyanin and the heme region of cytochrome f. Rigid-body structure calculations using NMR-derived restraints demonstrate that plastocyanin is oriented in a "head-on" fashion, with the long axis of the molecule perpendicular to the heme plane. Remarkably, the structure and affinity of the complex are independent of ionic strength, indicating that there is little electrostatic interaction. Lowering the pH results in limited reorganization of the complex interface, while the binding affinity is unaffected. Therefore, protonation of the exposed copper ligand, His92, plays only a minor role in the complex. In contrast to other electron-transfer complexes, the plastocyanin-cytochrome f complex from P. laminosum is predominantly controlled by hydrophobic interactions. These findings are discussed in the context of the previously characterized angiosperm complex.

Bacterial Proteins↗
PubMed2001

NMR structure of the LCCL domain and implications for DFNA9 deafness disorder.

The LCCL domain is a recently discovered, conserved protein module named after its presence in Limulus factor C, cochlear protein Coch-5b2 and late gestation lung protein Lgl1. The LCCL domain plays a key role in the autosomal dominant human deafness disorder DFNA9. Here we report the nuclear magnetic resonance (NMR) structure of the LCCL domain from human Coch-5b2, where dominant mutations leading to DFNA9 deafness disorder have been identified. The fold is novel. Four of the five known DFNA9 mutations are shown to involve at least partially solvent-exposed residues. Except for the Trp91Arg mutant, expression of these four LCCL mutants resulted in misfolded proteins. These results suggest that Trp91 participates in the interaction with a binding partner. The unexpected sensitivity of the fold with respect to mutations of solvent-accessible residues might be attributed to interference with the folding pathway of this disulfide-containing domain.

Amino Acid Sequence↗
PubMed2001

Thioredoxin fold as homodimerization module in the putative chaperone ERp29: NMR structures of the domains and experimental model of the 51 kDa dimer.

BACKGROUND: ERp29 is a ubiquitously expressed rat endoplasmic reticulum (ER) protein conserved in mammalian species. Fold predictions suggest the presence of a thioredoxin-like domain homologous to the a domain of human protein disulfide isomerase (PDI) and a helical domain similar to the C-terminal domain of P5-like PDIs. As ERp29 lacks the double-cysteine motif essential for PDI redox activity, it is suggested to play a role in protein maturation and/or secretion related to the chaperone function of PDI. ERp29 self-associates into 51 kDa dimers and also higher oligomers. RESULTS: 3D structures of the N- and C-terminal domains determined by NMR spectroscopy confirmed the thioredoxin fold for the N-terminal domain and yielded a novel all-helical fold for the C-terminal domain. Studies of the full-length protein revealed a short, flexible linker between the two domains, homodimerization by the N-terminal domain, and the presence of interaction sites for the formation of higher molecular weight oligomers. A gadolinium-based relaxation agent is shown to present a sensitive tool for the identification of macromolecular interfaces by NMR. CONCLUSIONS: ERp29 is the first eukaryotic PDI-related protein for which the structures of all domains have been determined. Furthermore, an experimental model of the full-length protein and its association states was established. It is the first example of a protein where the thioredoxin fold was found to act as a specific homodimerization module, without covalent linkages or supporting interactions by further domains. A homodimerization module similar as in ERp29 may also be present in homodimeric human PDI.

Amino Acid Sequence↗
PubMed2001

Sensitive 1H-31P correlations with 5' methylene protons of DNA via homonuclear double-quantum coherence.

A novel pulse sequence is presented for the correlation of 5' and 5'' protons in DNA with phosphorus. Double-quantum coherence between the methylene protons is used to generate 1H5'-31P and 1H5''-31P cross peaks in an HMQC-type experiment. The resolution for these cross peaks is significantly improved over that of conventional HSQC experiments, as cross peaks between 1H4' and 31P are largely suppressed and a 3D version of the experiment can be performed with little penalty in sensitivity. In addition, sensitivity is favoured by slower relaxation of the double-quantum coherence and a more favourable multiplet fine structure in the acquisition dimension.

DNA↗
PubMed2001

Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest.

The coordinated termination of DNA replication is an important step in the life cycle of bacteria with circular chromosomes, but has only been defined at a molecular level in two systems to date. Here we report the structure of an engineered replication terminator protein (RTP) of Bacillus subtilis in complex with a 21 base pair DNA by X-ray crystallography at 2.5 A resolution. We also use NMR spectroscopic titration techniques. This work reveals a novel DNA interaction involving a dimeric 'winged helix' domain protein that differs from predictions. While the two recognition helices of RTP are in close contact with the B-form DNA major grooves, the 'wings' and N-termini of RTP do not form intimate contacts with the DNA. This structure provides insight into the molecular basis of polar replication fork arrest based on a model of cooperative binding and differential binding affinities of RTP to the two adjacent binding sites in the complete terminator.

Amino Acid Motifs↗
PubMed2000

Ring opening of benzo[a]pyrene in the germ-free rat is a novel pathway for formation of potentially genotoxic metabolites.

The metabolism of benzo[a]pyrene (BP) is known to lead to a large number of oxygenated compounds, some of which can bind covalently to DNA. We have studied the integrated metabolism of BP in vivo in germ-free rats given (14)C-labeled BP. Urinary metabolites were separated into groups according to acidity using lipophilic ion exchangers. The groups were analyzed by mass spectrometry and were further fractionated by high-performance liquid chromatography. The fraction of urinary metabolites previously shown to contain N-acetylcysteine and glucuronic acid conjugates was found to contain derivatives of 7-oxo-benz[d]anthracene-3,4-dicarboxylic acid as major components. These compounds, which were identified by mass spectrometry and NMR, accounted for about 30% of the total metabolites in urine, demonstrating that, surprisingly, ring opening is a major pathway for metabolism of BP in the germ-free rat. The dicarboxylic acid may be excreted in urine as an ester glucuronide. By using the single cell gel electrophoresis or COMET assay, we were able to demonstrate that the anhydride of 7-oxo-benz[d]anthracene-3, 4-dicarboxylic acid was an efficient inducer of DNA damage. Taken together, these results indicate that the novel ring opening metabolic pathway may provide alternative mechanisms for the toxicity of BP.

Animals↗
PubMed2000

NMR structure of oxidized glutaredoxin 3 from Escherichia coli.

A high precision NMR structure of oxidized glutaredoxin 3 [C65Y] from Escherichia coli has been determined. The conformation of the active site including the disulphide bridge is highly similar to those in glutaredoxins from pig liver and T4 phage. A comparison with the previously determined structure of glutaredoxin 3 [C14S, C65Y] in a complex with glutathione reveals conformational changes between the free and substrate-bound form which includes the sidechain of the conserved, active site tyrosine residue. In the oxidized form this tyrosine is solvent exposed, while it adopts a less exposed conformation, stabilized by hydrogen bonds, in the mixed disulfide with glutathione. The structures further suggest that the formation of a covalent linkage between glutathione and glutaredoxin 3 is necessary in order to induce these structural changes upon binding of the glutathione peptide. This could explain the observed low affinity of glutaredoxins for S-blocked glutathione analogues, in spite of the fact that glutaredoxins are highly specific reductants of glutathione mixed disulfides.

Amino Acid Sequence↗
PubMed2000

Time-shared X(omega(1))-half-filter for improved sensitivity in subspectral editing.

Experiments with X-half-filter elements allow the separation of the resonances from protons bound and unbound to a spin X into different subspectra. This Communication presents a modified half-filter element where the filter delay is simultaneously used for chemical shift labeling and scalar coupling evolution in a semi-constant time experiment. The filter element is demonstrated with a (1)H NOESY spectrum of a 28.5-kDa 2:1 complex between the uniformly (13)C-labeled N-terminal domain of Escherichia coli arginine repressor and operator DNA.

Carbon Isotopes↗
PubMed2000

Preliminary X-ray crystallographic and NMR studies on the exonuclease domain of the epsilon subunit of Escherichia coli DNA polymerase III.

The structured core of the N-terminal 3'-5' exonuclease domain of epsilon, the proofreading subunit of Escherichia coli DNA polymerase III, was defined by multidimensional NMR experiments with uniformly (15)N-labeled protein: it comprises residues between Ile-4 and Gln-181. A 185-residue fragment, termed epsilon(1-185), was crystallized by the hanging drop vapor diffusion method in the presence of thymidine-5'-monophosphate, a product inhibitor, and Mn(2+) at pH 5.8. The crystals are tetragonal, with typical dimensions 0.2 mm x 0.2 mm x 1.0 mm, grow over about 2 weeks at 4 degrees C, and diffract X-rays to 2.0 A. The space group was determined to be P4(n)2(1)2 (n = 0, 1, 2, 3), with unit cell dimensions a = 60.8 A, c = 111.4 A.

Catalytic Domain↗
PubMed2000

Sensitivity enhancement in (HCA)CONH experiments.

A novel sensitivity-enhancement technique is proposed for experiments which correlate protein backbone resonances and start with magnetization from 13Calpha-1Halpha groups. The technique is based on replenishing magnetization lost by dipole-CSA cross-correlated relaxation of the 13Calpha spin with 13Calpha steady state magnetization. The principle is demonstrated for the (HCA)CONH experiment, resulting in 1.6-fold sensitivity enhancement compared to the HN(CA)CO experiment. Furthermore, other versions of the (HCA)CONH experiment were evaluated, including a novel experiment with spin-locking of transverse 13C-1H two-spin coherence, and a cross-correlation compensated (CA)CONH experiment which starts from 13C rather than 1H magnetization.

Bacterial Proteins↗
PubMed2000

NMR experiments for the sign determination of homonuclear scalar and residual dipolar couplings.

A modified version of the JHH-TOCSY experiment, 'signed COSY', is presented that allows the determination of the sign of residual dipolar 1H-1H coupling constants with respect to the sign of one-bond 1H-X coupling constants in linear three-spin systems X-1H-1H, where X = 13C or 15N. In contrast to the original JHH-TOCSY experiments, the signs of J(HH) couplings may be determined for CH2-CH2 moieties and for uniformly 13C/15N-labelled samples. In addition, sensitivity is enhanced, diagonal peaks are suppressed and cross peaks are observed only between directly coupled protons, as in a COSY spectrum.

Aprotinin↗
PubMed1999

Molecular electroporation: a unifying concept for the description of membrane pore formation by antibacterial peptides, exemplified with NK-lysin.

The antibacterial activity of many small, positively charged peptides and proteins is based on pore formation in lipid bilayers. It is here proposed to arise from an electroporation effect. This hypothesis is supported by calculations of the electrostatic potential of NK-lysin associated to a membrane. For a significant area of the protein-membrane interface, the electrostatic potential is found to be above the minimum threshold for electroporation. A single highly charged alpha-helical segment of NK-lysin is mainly responsible for this effect. It is experimentally demonstrated that a peptide comprising this helix has antibacterial activity. We propose that superficial association to membranes suffices to trigger electroporation, provided the peptide is sufficiently charged. The effect is referred to as molecular electroporation.

Anti-Bacterial Agents↗
PubMed1999

NMR structure of the N-terminal domain of E. coli DnaB helicase: implications for structure rearrangements in the helicase hexamer.

BACKGROUND: DnaB is the primary replicative helicase in Escherichia coli. Native DnaB is a hexamer of identical subunits, each consisting of a larger C-terminal domain and a smaller N-terminal domain. Electron-microscopy data show hexamers with C6 or C3 symmetry, indicating large domain movements and reversible pairwise association. RESULTS: The three-dimensional structure of the N-terminal domain of E. coli DnaB was determined by nuclear magnetic resonance (NMR) spectroscopy. Structural similarity was found with the primary dimerisation domain of a topoisomerase, the gyrase A subunit from E. coli. A monomer-dimer equilibrium was observed for the isolated N-terminal domain of DnaB. A dimer model with C2 symmetry was derived from intermolecular nuclear Overhauser effects, which is consistent with all available NMR data. CONCLUSIONS: The monomer-dimer equilibrium observed for the N-terminal domain of DnaB is likely to be of functional significance for helicase activity, by participating in the switch between C6 and C3 symmetry of the helicase hexamer.

Amino Acid Sequence↗
PubMed1999

Direct NMR observation of the Cys-14 thiol proton of reduced Escherichia coli glutaredoxin-3 supports the presence of an active site thiol-thiolate hydrogen bond.

The active site of Escherichia coli glutaredoxin-3 (Grx3) consists of two redox active cysteine residues in the sequence -C11-P-Y-C14-H-. The 1H NMR resonance of the cysteine thiol proton of Cys-14 in reduced Grx3 is observed at 7.6 ppm. The large downfield shift and NOEs observed with this thiol proton resonance suggest the presence of a hydrogen bond with the Cys-11 thiolate, which is shown to have an abnormally low pKa value. A hydrogen bond would also agree with activity data of Grx3 active site mutants. Furthermore, the activity is reduced in a Grx3 H15V mutant, indicating electrostatic contributions to the stabilization of the Cys-11 thiolate.

Bacterial Proteins↗
PubMed1999

NMR structure of Escherichia coli glutaredoxin 3-glutathione mixed disulfide complex: implications for the enzymatic mechanism.

Glutaredoxins (Grxs) catalyze reversible oxidation/reduction of protein disulfide groups and glutathione-containing mixed disulfide groups via an active site Grx-glutathione mixed disulfide (Grx-SG) intermediate. The NMR solution structure of the Escherichia coli Grx3 mixed disulfide with glutathione (Grx3-SG) was determined using a C14S mutant which traps this intermediate in the redox reaction. The structure contains a thioredoxin fold, with a well-defined binding site for glutathione which involves two intermolecular backbone-backbone hydrogen bonds forming an antiparallel intermolecular beta-bridge between the protein and glutathione. The solution structure of E. coli Grx3-SG also suggests a binding site for a second glutathione in the reduction of the Grx3-SG intermediate, which is consistent with the specificity of reduction observed in Grxs. Molecular details of the structure in relation to the stability of the intermediate and the activity of Grx3 as a reductant of glutathione mixed disulfide groups are discussed. A comparison of glutathione binding in Grx3-SG and ligand binding in other members of the thioredoxin superfamily is presented, which illustrates the highly conserved intermolecular interactions in this protein family.

Amino Acid Sequence↗
PubMed1999

Selection of a peptide ligand to the p75 neurotrophin receptor death domain and determination of its binding sites by NMR.

The p75 neurotrophin receptor (p75(NTR)) contains a conserved death domain module similar to that of the cytotoxic receptors Fas and TNFR-1. Here, we describe the selection of peptide ligands from a combinatorial library using a variation of the selectively-infective phage (SIP) method directed to the death domain of p75(NTR). The binding sites on the death domain of p75(NTR) were identified for a 15 amino acid residue peptide by nuclear magnetic resonance (NMR) spectroscopy. The selected peptides may be useful for probing the function of the p75(NTR) death domain and aid in defining its downstream signalling mechanism.

Amino Acid Sequence↗
PubMed1999

Measurement of Magnitude and Sign of Heteronuclear Coupling Constants in Transition Metal Complexes.

Sets of specifically tailored E.COSY-type correlation experiments and double-quantum/zero-quantum (DQ/ZQ) experiments are presented which enable the determination of sign and size of small heteronuclear coupling constants across the metal center of transition metal complexes. For the octahedrally coordinated complexes, [Ru(TPM)(H)(CO)(PPh3)]+[BF4]- (1) and [Ir(TPM)(H)(CO)(CO2CH3)]+[BF4]- (2), 14 of 15 and 15 of 15 possible two-bond scalar coupling constants across the metal center were measured, respectively, using 15N and 15N/13C enriched samples (TPM = tris(1-pyrazolyl)methane)). The reduced coupling constants 2KX-M-Y = 4pi2 2J/(hgammaXgammaY) were found to be positive when the coupled nuclei X and Y were trans with respect to the metal center, and negative when the coupled nuclei were in cis position. The validity of this sign rule was verified for JCC, JNN, JPN, JPC, JCN, JHP, JHC, and JHN couplings. Idiosyncracies associated with 2D NMR spectra for the sign determination of coupling constants with 15N which lead to corrections for the signs of JHN, JPN, and JCN couplings reported previously are discussed. Copyright 1999 Academic Press.

Journal Article↗