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K Wüthrich

Publications and source records attributed to K Wüthrich.

At least 19 recordsLinked to original sources
PubMed1999

Polarization transfer by cross-correlated relaxation in solution NMR with very large molecules.

In common multidimensional NMR experiments for studies of biological macromolecules in solution, magnetization transfers via spin-spin couplings [insensitive nuclei enhanced by polarization transfer (INEPT)] are key elements of the pulse schemes. For molecular weights beyond 100,000, transverse relaxation during the transfer time may become a limiting factor. This paper presents a transfer technique for work with big molecules, cross relaxation-enhanced polarization transfer (CRINEPT), which largely reduces the size limitation of INEPT transfers with the use of cross-correlated relaxation-induced polarization transfer. The rate of polarization transfer by cross-correlated relaxation is proportional to the rotational correlation time, so that it becomes a highly efficient transfer mechanism for solution NMR with very high molecular weights. As a first implementation, [15N,1H]-correlation experiments were designed that make use of cross-correlation between dipole-dipole coupling and chemical shift anisotropy of the 15N---1H-moieties for both CRINEPT and transverse relaxation-optimized spectroscopy (TROSY). When compared with INEPT-based [15N,1H]-TROSY, these experiments yielded up to 3-fold signal enhancement for amide groups of a 110,000-Da protein in aqueous solution at 4 degrees C. CRINEPT opens avenues for solution NMR with supramolecular structures such as membrane proteins solubilized in micelles or lipid vesicles, proteins attached to nucleic acid fragments, or oligomeric proteins.

Energy Transfer↗
PubMed1999

NMR structure of the human oncofoetal fibronectin ED-B domain, a specific marker for angiogenesis.

BACKGROUND: The process of angiogenesis (i.e. the formation of new blood vessels from pre-existing ones) is fundamental to physiological processes such as reproduction, development and repair, as well as to pathological conditions such as tumor progression, rheumathoid arthritis and ocular disorders. The oncofoetal ED-B domain, a specific marker of angiogenesis, consists of 91 amino acid residues that are inserted by alternative splicing into the fibronectin (FN) molecule. RESULTS: The NMR structure of the ED-B domain is reported and reveals important differences from other FN type III domains. A comparison of the ED-B domain with the crystal structure of a four-domain FN fragment shows the novel features of ED-B to be located in loop regions that are buried at interdomain interfaces, and which therefore largely determine the global shape of the FN molecule. The negatively charged amino acids in this highly acidic protein are uniformly distributed over the molecular surface, with the sole exception of a solvent-exposed hydrophobic patch that represents a potential specific recognition site. Epitope mapping with 82 decapeptides that span the ED-B sequence revealed that three ED-B-specific monoclonal antibodies, which selectively target newly forming blood vessels in tumor-bearing mice, bind to adjacent regions on the ED-B surface. CONCLUSIONS: The NMR structure enables the identification of a large surface area of the ED-B domain that appears to be accessible in vivo, opening up new diagnostic and therapeutic opportunities. Furthermore, the mapping of specific monoclonal antibodies to the three-dimensional structure of the ED-B domain, and their use in angiogenesis inhibition experiments, provides a basis for further investigation of the role of the ED-B domain in the formation of new blood vessels.

Amino Acid Sequence↗
PubMed1999

Pilus chaperone FimC-adhesin FimH interactions mapped by TROSY-NMR.

The 23 kDa two-domain periplasmic chaperone FimC from Escherichia coli is required for the assembly of type-1 pili, which are filamentous, highly oligomeric protein complexes anchored to the outer bacterial membrane that mediate adhesion of pathogenic E. coli strains to host cell surfaces. Here we identified the contact sites on the surface of the NMR structure of FimC that are responsible for the binding of the 28 kDa mannose-binding type-1 pilus subunit FimH by 15N and 1H NMR chemical shift mapping, using transverse relaxation-optimized spectroscopy (TROSY). The FimH-binding surface of FimC is formed nearly entirely by the N-terminal domain, and its extent and shape indicate that FimC binds a folded form of the pilus subunits.

Adhesins, Bacterial↗
PubMed1999

NMR studies of subunit c of the ATP synthase from Propionigenium modestum in dodecylsulphate micelles.

The structure of the Na+, Li+ or H+-binding c subunit of the ATP synthase from Propionigenium modestum was studied by NMR. Subunit c in dodecylsulphate micelles consists of four alpha-helical segments, I-IV, that are connected by short linker peptides with non-regular secondary structures. We propose that helices I (V4-I26) and IV (I69-V85) are membrane-spanning structures, and that helices II and III and the intervening hydrophilic loop are located in the cytoplasm. The Na+-binding residues Q32, E65 and S66 are located in the I-->II and III-->IV helix connections, probably near the membrane surface on the cytoplasmic side.

Amino Acid Sequence↗
PubMed1999

Amino acid biosynthesis in the halophilic archaeon Haloarcula hispanica.

Biosynthesis of proteinogenic amino acids in the extremely halophilic archaeon Haloarcula hispanica was explored by using biosynthetically directed fractional 13C labeling with a mixture of 90% unlabeled and 10% uniformly 13C-labeled glycerol. The resulting 13C-labeling patterns in the amino acids were analyzed by two-dimensional 13C,1H correlation spectroscopy. The experimental data provided evidence for a split pathway for isoleucine biosynthesis, with 56% of the total Ile originating from threonine and pyruvate via the threonine pathway and 44% originating from pyruvate and acetyl coenzyme A via the pyruvate pathway. In addition, the diaminopimelate pathway involving diaminopimelate dehydrogenase was shown to lead to lysine biosynthesis and an analysis of the 13C-labeling pattern in tyrosine indicated novel biosynthetic pathways that have so far not been further characterized. For the 17 other proteinogenic amino acids, the data were consistent with data for commonly found biosynthetic pathways. A comparison of our data with the amino acid metabolisms of eucarya and bacteria supports the theory that pathways for synthesis of proteinogenic amino acids were established before ancient cells diverged into archaea, bacteria, and eucarya.

Acetyl Coenzyme A↗
PubMed1998

NMR scalar couplings across Watson-Crick base pair hydrogen bonds in DNA observed by transverse relaxation-optimized spectroscopy.

This paper describes the NMR observation of 15N---15N and 1H---15N scalar couplings across the hydrogen bonds in Watson-Crick base pairs in a DNA duplex, hJNN and hJHN. These couplings represent new parameters of interest for both structural studies of DNA and theoretical investigations into the nature of the hydrogen bonds. Two dimensional [15N,1H]-transverse relaxation-optimized spectroscopy (TROSY) with a 15N-labeled 14-mer DNA duplex was used to measure hJNN, which is in the range 6-7 Hz, and the two-dimensional hJNN-correlation-[15N,1H]-TROSY experiment was used to correlate the chemical shifts of pairs of hydrogen bond-related 15N spins and to observe, for the first time, hJHN scalar couplings, with values in the range 2-3.6 Hz. TROSY-based studies of scalar couplings across hydrogen bonds should be applicable for large molecular sizes, including protein-bound nucleic acids.

Base Pairing↗
PubMed1998

TROSY in triple-resonance experiments: new perspectives for sequential NMR assignment of large proteins.

The NMR assignment of 13C, 15N-labeled proteins with the use of triple resonance experiments is limited to molecular weights below approximately 25,000 Daltons, mainly because of low sensitivity due to rapid transverse nuclear spin relaxation during the evolution and recording periods. For experiments that exclusively correlate the amide proton (1HN), the amide nitrogen (15N), and 13C atoms, this size limit has been previously extended by additional labeling with deuterium (2H). The present paper shows that the implementation of transverse relaxation-optimized spectroscopy ([15N,1H]-TROSY) into triple resonance experiments results in several-fold improved sensitivity for 2H/13C/15N-labeled proteins and approximately twofold sensitivity gain for 13C/15N-labeled proteins. Pulse schemes and spectra recorded with deuterated and protonated proteins are presented for the [15N, 1H]-TROSY-HNCA and [15N, 1H]-TROSY-HNCO experiments. A theoretical analysis of the HNCA experiment shows that the primary TROSY effect is on the transverse relaxation of 15N, which is only little affected by deuteration, and predicts sensitivity enhancements that are in close agreement with the experimental data.

Animals↗
PubMed1998

Prion protein NMR structure and familial human spongiform encephalopathies.

The refined NMR structure of the mouse prion protein domain mPrP(121-231) and the recently reported NMR structure of the complete 208-residue polypeptide chain of mPrP are used to investigate the structural basis of inherited human transmissible spongiform encephalopathies. In the cellular form of mPrP no spatial clustering of mutation sites is observed that would indicate the existence of disease-specific subdomains. A hydrogen bond between residues 128 and 178 provides a structural basis for the observed highly specific influence of a polymorphism in position 129 in human PrP on the disease phenotype that segregates with the mutation Asp-178-Asn. Overall, the NMR structure implies that only part of the disease-related amino acid replacements lead to reduced stability of the cellular form of PrP, indicating that subtle structural differences in the mutant proteins may affect intermolecular signaling in a variety of different ways.

Animals↗
PubMed1998

Recommendations for the presentation of NMR structures of proteins and nucleic acids--IUPAC-IUBMB-IUPAB Inter-Union Task Group on the standardization of data bases of protein and nucleic acid structures determined by NMR spectroscopy.

The recommendations presented here are designed to support easier communication of NMR data and NMR structures of proteins and nucleic acids through unified nomenclature and reporting standards. Much of this document pertains to the reporting of data in journal articles; however, in the interest of the future development of structural biology, it is desirable that the bulk of the reported information be stored in computer-accessible form and be freely accessible to the scientific community in standardized formats for data exchange. These recommendations stem from an IUPAC-IUBMB-IUPAB inter-union venture with the direct involvement of ICSU and CODATA. The Task Group has reviewed previous formal recommendations and has extended them in the light of more recent developments in the field of biomolecular NMR spectroscopy. Drafts of the recommendations presented here have been examined critically by more than 50 specialists in the field and have gone through two rounds of extensive modification to incorporate suggestions and criticisms.

Amino Acid Sequence↗
PubMed1998

Recommendations for the presentation of NMR structures of proteins and nucleic acids.

The recommendations presented here are designed to support easier communication of NMR data and NMR structures of proteins and nucleic acids through unified nomenclature and reporting standards. Much of this document pertains to the reporting of data in journal articles; however, in the interest of the future development of structural biology, it is desirable that the bulk of the reported information be stored in computer-accessible form and be freely accessible to the scientific community in standardized formats for data exchange. These recommendations stem from an IUPAC-IUBMB-IUPAB inter-union venture with the direct involvement of ICSU and CODATA. The Task Group has reviewed previous formal recommendations and has extended them in the light of more recent developments in the field of biomolecular NMR spectroscopy. Drafts of the recommendations presented here have been examined critically by more than 50 specialists in the field and have gone through two rounds of extensive modification to incorporate suggestions and criticisms.

Databases as Topic↗
PubMed1998

Prion protein structural features indicate possible relations to signal peptidases.

Transmissible spongiform encephalopathies (TSEs) in mammalian species are believed to be caused by an oligomeric isoform, PrP(Sc), of the cellular prion protein, PrP(C). One of the key questions in TSE research is how the observed accumulation of PrP(Sc), or possibly the concomitant depletion of PrP(C) can cause fatal brain damage. Elucidation of the so far unknown function of PrP(C) is therefore of crucial importance. PrP(C) is a membrane-anchored cell surface protein that possesses a so far unique three-dimensional structure. While the N-terminal segment 23-120 of PrP(C) is flexibly disordered, its C-terminal residues 121-231 form a globular domain with three alpha-helices and a two-stranded beta-sheet. Here we report the observation of structural similarities between the domain of PrP(121-231) and the soluble domains of membrane-anchored signal peptidases. At the level of the primary structure we find 23% identity and 41% similarity between residues 121-217 of the C-terminal domain of murine PrP and a catalytic domain of the rat signal peptidase. The invariant PrP residues Tyr-128 and His-177 align with the two presumed active-site residues of signal peptidases and are in close spatial proximity in the three-dimensional structure of PrP(121-231).

Animals↗
PubMed1998

Structure comparison of human glioma pathogenesis-related protein GliPR and the plant pathogenesis-related protein P14a indicates a functional link between the human immune system and a plant defense system.

The human glioma pathogenesis-related protein (GliPR) is highly expressed in the brain tumor glioblastoma multiforme and exhibits 35% amino acid sequence identity with the tomato pathogenesis-related (PR) protein P14a, which has an important role for the plant defense system. A molecular model of GliPR was computed with the distance geometry program DIANA on the basis of a P14a-GliPR sequence alignment and a set of 1,200 experimental NMR conformational constraints collected with P14a. The GliPR structure is represented by a group of 20 conformers with small residual DIANA target function values, low AMBER-energies after restrained energy-minimization with the program OPAL, and an average rms deviation relative to the mean of 1.6 A for the backbone heavy atoms. Comparison of the GliPR model with the P14a structure lead to the identification of a common partially solvent-exposed spatial cluster of four amino acid residues, His-69, Glu-88, Glu-110, and His-127 in the GliPR numeration. This cluster is conserved in all known plant PR proteins of class 1, indicating a common putative active site for GliPR and PR-1 proteins and thus a functional link between the human immune system and a plant defense system.

Amino Acid Sequence↗
PubMed1998

Dynamic modes of the flipped-out cytosine during HhaI methyltransferase-DNA interactions in solution.

Flipping of a nucleotide out of a B-DNA helix into the active site of an enzyme has been observed for the HhaI and HaeIII cytosine-5 methyltransferases (M.HhaI and M.HaeIII) and for numerous DNA repair enzymes. Here we studied the base flipping motions in the binary M. HhaI-DNA and the ternary M.HhaI-DNA-cofactor systems in solution. Two 5-fluorocytosines were introduced into the DNA in the places of the target cytosine and, as an internal control, a cytosine positioned two nucleotides upstream of the recognition sequence 5'-GCGC-3'. The 19F NMR spectra combined with gel mobility data show that interaction with the enzyme induces partition of the target base among three states, i.e. stacked in the B-DNA, an ensemble of flipped-out forms and the flipped-out form locked in the enzyme active site. Addition of the cofactor analogue S-adenosyl-L-homocysteine greatly enhances the trapping of the target cytosine in the catalytic site. Distinct dynamic modes of the target cytosine have thus been identified along the reaction pathway, which includes novel base-flipping intermediates that were not observed in previous X-ray structures. The new data indicate that flipping of the target base out of the DNA helix is not dependent on binding of the cytosine in the catalytic pocket of M.HhaI, and suggest an active role of the enzyme in the opening of the DNA duplex.

Base Composition↗
PubMed1998

Pulmonary surfactant-associated polypeptide C in a mixed organic solvent transforms from a monomeric alpha-helical state into insoluble beta-sheet aggregates.

In the 35-residue pulmonary surfactant-associated lipopolypeptide C (SP-C), the stability of the valyl-rich alpha-helix comprising residues 9-34 has been monitored by circular dichroism, nuclear magnetic resonance, and Fourier transform infrared spectroscopy in both a mixed organic solvent and in phospholipid micelles. The alpha-helical form of SP-C observed in freshly prepared solutions in a mixed solvent of CHCl3/CH3OH/0.1 M HCl 32:64:5 (v/v/v) at 10 degrees C undergoes within a few days an irreversible transformation to an insoluble aggregate that contains beta-sheet secondary structure. Hydrogen exchange experiments revealed that this conformational transition proceeds through a transition state with an Eyring free activation enthalpy of about 100 kJ mol(-1), in which the polypeptide segment 9-27 largely retains a helical conformation. In dodecylphosphocholine micelles, the helical form of SP-C was maintained after seven weeks at 50 degrees C. The alpha-helical form of SP-C thus seems to be the thermodynamically most stable state in this micellar environment, whereas its presence in freshly prepared samples in the aforementioned mixed solvent is due to a high kinetic barrier for unfolding. These observations support a previously proposed pathway for in vivo synthesis of SP-C through proteolytic processing from a 21-kDa precursor protein.

Amino Acid Sequence↗
PubMed1998

Solvent magnetization artifacts in high-field NMR studies of macromolecular hydration.

With the use of high magnetic fields and improved quality factor ratings of the probeheads in modern NMR spectrometers, radiation damping becomes more and more important. In addition, the demagnetizing field effect from protonated solvents gains significance with the increase of the magnetic field strength. During a typical NMR pulse sequence the magnetic fields caused by these effects become time-dependent, which makes the system nonlinear and may, for example, measurably influence the precession frequencies of all nuclei in the sample. Since radiation damping can affect signals that are several kilohertz away from the solvent resonance, the amplitude, phase, and frequency of the desired signals can be disturbed so as to give rise to spectral artifacts. In particular when difference methods are used to obtain the final spectrum, the data sets may be severely deteriorated by such artifacts. This paper investigates effects from the demagnetizing field and from radiation damping with a selection of pulse sequences in use for studies of macromolecular hydration, and strategies are described for the detection and elimination of the ensuing artifacts.

Artifacts↗
PubMed1998

Automated peak picking and peak integration in macromolecular NMR spectra using AUTOPSY.

A new approach for automated peak picking of multidimensional protein NMR spectra with strong overlap is introduced, which makes use of the program AUTOPSY (automated peak picking for NMR spectroscopy). The main elements of this program are a novel function for local noise level calculation, the use of symmetry considerations, and the use of lineshapes extracted from well-separated peaks for resolving groups of strongly overlapping peaks. The algorithm generates peak lists with precise chemical shift and integral intensities, and a reliability measure for the recognition of each peak. The results of automated peak picking of NOESY spectra with AUTOPSY were tested in combination with the combined automated NOESY cross peak assignment and structure calculation routine NOAH implemented in the program DYANA. The quality of the resulting structures was found to be comparable with those from corresponding data obtained with manual peak picking.

Algorithms↗
PubMed1998

In pre-sterol carrier protein 2 (SCP2) in solution the leader peptide 1-20 is flexibly disordered, and residues 21-143 adopt the same globular fold as in mature SCP2.

The preform of the rabbit sterol carrier protein 2 (pre-rSCP2) was cloned, the uniformly 15N-labelled protein expressed in Escherichia coli and studied by three-dimensional 15N-resolved nuclear magnetic resonance spectroscopy. In spite of its low solubility in aqueous solution of only approximately 0.3 mM, sequential 15N and 1H backbone resonance assignments were obtained for 105 out of the 143 residues. From comparison of the sequential and medium-range nuclear Overhauser effects (NOEs) in the two proteins, all regular secondary structures previously determined in mature human SCP2 (hSCP2) [Szyperski et al. (1993) FEBS Lett. 335: 18-26] were also identified in pre-rSCP2. Near-identity of the backbone 15N and 1H chemical shifts and 1:1 correspondence of 24 long-range NOEs to backbone amide groups in the two proteins show that the residues 21-143 adopt the same globular fold in pre-rSCP2 and mature hSCP2. The N-terminal 20-residue leader peptide of pre-rSCP2 is flexibly disordered in solution and does not observably affect the conformation of the polypeptide segment 21-143.

Amino Acid Sequence↗