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K Wüthrich

Publications and source records attributed to K Wüthrich.

At least 37 records · Page 2Linked to original sources
PubMed2000

NMR structures of three single-residue variants of the human prion protein.

The NMR structures of three single-amino acid variants of the C-terminal domain of the human prion protein, hPrP(121-230), are presented. In hPrP(M166V) and hPrP(R220K) the substitution is with the corresponding residue in murine PrP, and in hPrP(S170N) it is with the corresponding Syrian hamster residue. All three substitutions are in the surface region of the structure of the cellular form of PrP (PrP(C)) that is formed by the C-terminal part of helix 3, with residues 218-230, and a loop of residues 166-172. This molecular region shows high species variability and has been implicated in specific interactions with a so far not further characterized "protein X," and it is related to the species barrier for transmission of prion diseases. As expected, the three variant hPrP(121-230) structures have the same global architecture as the previously determined wild-type bovine, human, murine, and Syrian hamster prion proteins, but with the present study two localized "conformational markers" could be related with single amino acid exchanges. These are the length and quality of definition of helix 3, and the NMR-observability of the residues in the loop 166-172. Poor definition of the C-terminal part of helix 3 is characteristic for murine PrP and has now been observed also for hPrP(R220K), and NMR observation of the complete loop 166-172 has so far been unique for Syrian hamster PrP and is now also documented for hPrP(S170N).

Amino Acid Sequence↗
PubMed2000

NMR structure of the sterol carrier protein-2: implications for the biological role.

The determination of the NMR structure of the sterol carrier protein-2 (SCP2), analysis of backbone (15)N spin relaxation parameters and NMR studies of nitroxide spin-labeled substrate binding are presented as a new basis for investigations of the mode of action of SCP2. The SCP2 fold is formed by a five-stranded beta-sheet and four alpha-helices. Fatty acid binding to a hydrophobic surface area formed by amino acid residues of the first and third helices, and the beta-sheet, which are all located in the polypeptide segment 8-102, was identified with the use of the spin-labeled substrate 16-doxylstearic acid. In the free protein, the lipid-binding site is covered by the C-terminal segment 105-123, suggesting that this polypeptide segment, which carries the peroxisomal targeting signal (PTS1), might be involved in the regulation of ligand binding.

Animals↗
PubMed2000

NMR solution structure of the human prion protein.

The NMR structures of the recombinant human prion protein, hPrP(23-230), and two C-terminal fragments, hPrP(90-230) and hPrP(121-230), include a globular domain extending from residues 125-228, for which a detailed structure was obtained, and an N-terminal flexibly disordered "tail." The globular domain contains three alpha-helices comprising the residues 144-154, 173-194, and 200-228 and a short anti-parallel beta-sheet comprising the residues 128-131 and 161-164. Within the globular domain, three polypeptide segments show increased structural disorder: i.e., a loop of residues 167-171, the residues 187-194 at the end of helix 2, and the residues 219-228 in the C-terminal part of helix 3. The local conformational state of the polypeptide segments 187-193 in helix 2 and 219-226 in helix 3 is measurably influenced by the length of the N-terminal tail, with the helical states being most highly populated in hPrP(23-230). When compared with the previously reported structures of the murine and Syrian hamster prion proteins, the length of helix 3 coincides more closely with that in the Syrian hamster protein whereas the disordered loop 167-171 is shared with murine PrP. These species variations of local structure are in a surface area of the cellular form of PrP that has previously been implicated in intermolecular interactions related both to the species barrier for infectious transmission of prion disease and to immune reactions.

Animals↗
PubMed2000

The prion protein globular domain and disease-related mutants studied by molecular dynamics simulations.

In humans, familial forms of transmissible spongiform encephalopathies (TSE; "prion diseases") have been shown to segregate with the exchange of individual amino acids in the prion protein (PrP) sequence. We used the NMR structure of the globular domain of mouse PrP in the cellular form (PrP(C)) as a starting point for investigations by long-time molecular dynamics (MD) simulations at ambient temperature of likely impacts of such mutations on the PrP(C) structure, making use of the fact that species-related amino acid replacements between mouse PrP and human PrP are spatially well separated from the disease-related mutations in human PrP. In the MD simulations these amino acid substitutions were found to have a variety of different effects on the protein structure, with some species showing altered packing of regular secondary structure elements, while other mutants showed no or only strictly localized changes of the structure near the variant amino acid. The fact that some of the disease-related amino acid exchanges cause no measurable change of the PrP(C) structure indicates that their influence on the conformational transition to the scrapie form of PrP may be due to modified intermolecular interactions during the aggregation process.

Amino Acid Substitution↗
PubMed2000

TROSY and CRINEPT: NMR with large molecular and supramolecular structures in solution.

TROSY and CRINEPT are new techniques for solution NMR studies of molecular and supramolecular structures. They allow the collection of high-resolution spectra of structures with molecular weights >100 kDa, significantly extending the range of macromolecular systems that can be studied by NMR in solution. TROSY has already been used to map protein-protein interfaces, to conduct structural studies on membrane proteins and to study nucleic acid conformations in multimolecular assemblies. These techniques will help us to investigate the conformational states of individual macromolecular components and will support de novo protein structure determination in large supramolecular structures.

Aldehyde-Lyases↗
PubMed2000

NMR experiments for resonance assignments of 13C, 15N doubly-labeled flexible polypeptides: application to the human prion protein hPrP(23-230).

A combination of three heteronuclear three-dimensional NMR experiments tailored for sequential resonance assignments in uniformly 15N, 13C-labeled flexible polypeptide chains is described. The 3D (H)N(CO-TOCSY)NH, 3D (H)CA(CO-TOCSY)NH and 3D (H)CBCA(CO-TOCSY)NH schemes make use of the favorable 15N chemical shift dispersion in unfolded polypeptides, exploit the slow transverse 15N relaxation rates of unfolded polypeptides in high resolution constant-time [1H, 15N]-correlation experiments, and use carbonyl carbon homonuclear isotropic mixing to transfer magnetization sequentially along the amino acid sequence. Practical applications are demonstrated with the 100-residue flexible tail of the recombinant human prion protein, making use of spectral resolution up to 0.6 Hz in the 15N dimension, simultaneous correlation with the two adjacent amino acid residues to overcome problems associated with spectral overlap, and the potential of the presently described experiments to establish nearest-neighbor correlations across proline residues in the amino acid sequence.

Carbon Isotopes↗
PubMed2000

Sequence-specific NMR assignment of proteins by global fragment mapping with the program MAPPER.

A new program, MAPPER, for semiautomatic sequence-specific NMR assignment in proteins is introduced. The program uses an input of short fragments of sequentially neighboring residues, which have been assembled based on sequential NMR connectivities and for which either the 13C(alpha) and 13C(beta) chemical shifts or data on the amino acid type from other sources are known. MAPPER then performs an exhaustive search for self-consistent simultaneous mappings of all these fragments onto the protein sequence. Compared to using only the individual mappings of the spectroscopically connected fragments, the global mapping adds a powerful new constraint, which results in resolving many otherwise intractable ambiguities. In an initial application, virtually complete sequence-specific assignments were obtained for a 110 kDa homooctameric protein, 7,8-dihydroneopterin aldolase from Staphylococcus aureus.

Aldehyde-Lyases↗
PubMed2000

Deuterium isotope effects on the central carbon metabolism of Escherichia coli cells grown on a D2O-containing minimal medium.

Isotope effects on the central carbon metabolism due to the addition of variable amounts of D2O (0 to 70%) were investigated with biosynthetically directed fractional 13C-labeling for Escherichia coli BL21(DE3) cells during exponential growth on a M9 minimal medium containing a mixture of 70% unlabeled and 30% uniformly 13C-labeled glucose as the sole carbon source. The resulting 13C-labeling patterns in the amino acids were analysed by two-dimensional [13C,1H]-correlation spectroscopy. With the aforementioned growth conditions, higher D2O contents resulted in an increase of the anaplerotic supply of the tricarboxylic acid cycle via carboxylation of phosphoenolpyruvate when compared to the influx of acetyl-CoA. Furthermore, the addition of D2O affected the C1 metabolic pathways that involve Ser and Gly. Otherwise the E. coli cells showed identical topologies of the active biosynthetic pathways in H2O and at elevated D2O contents, and the metabolic flux ratios characterizing glycolysis and the pentose phosphate pathway were not measurably affected by the addition of D2O. Cells that had been adapted for growth in D2O exhibited the same response to the presence of D2O in the nutrient medium as non-adapted cells. Implications of these data for the preparation of recombinant deuterated proteins for NMR studies are discussed.

Carbon↗
PubMed2000

Determination of h2J(NN) and h1J(HN) coupling constants across Watson-Crick base pairs in the Antennapedia homeodomain-DNA complex using TROSY.

This paper describes NMR measurements of 15N-15N and 1H-15N scalar couplings across hydrogen bonds in Watson-Crick base pairs, h2J(NN) and h1J(HN), in a 17 kDa Antennapedia homeodomain-DNA complex. A new NMR experiment is introduced which relies on zero-quantum coherence-based transverse relaxation-optimized spectroscopy (ZQ-TROSY) and enables measurements of h1J(HN) couplings in larger molecules. The h2JNN and h1J(HN) couplings open a new avenue for comparative studies of DNA duplexes and other forms of nucleic acids free in solution and in complexes with proteins, drugs or possibly other classes of compounds.

Antennapedia Homeodomain Protein↗
PubMed2000

[15N,1H]/[13C,1H]-TROSY for simultaneous detection of backbone 15N-1H, aromatic 13C-1H and side-chain 15N-1H2 correlations in large proteins.

This paper describes a [15N,1H]/[13C,1H]-TROSY experiment for the simultaneous acquisition of the heteronuclear chemical shift correlations of backbone amide 15N-1H groups, side chain 15N-1H2 groups and aromatic 13C-1H groups in otherwise highly deuterated proteins. The 15N-1H and 13C-1H correlations are extracted from two subspectra of the same data set, thus preventing possible spectral overlap of aromatic and amide protons in the 1H dimension. The side-chain 15N-1H2 groups, which are suppressed in conventional [15N,1H)-TROSY, are observed with high sensitivity in the 15N-1H subspectrum. [15N,1H]/[13C,1H]-TROSY was used as the heteronuclear correlation block in a 3D [1H,1H]-NOESY-[15N,1H]/[13C,1H]-TROSY experiment with the membrane protein OmpA reconstituted in detergent micelles of molecular weight 80000 Da, which enabled the detection of numerous NOEs between backbone amide protons and both aromatic protons and side chain 15N-1H2 groups.

Bacterial Outer Membrane Proteins↗
PubMed2000

NMR characterization of a pH-dependent equilibrium between two folded solution conformations of the pheromone-binding protein from Bombyx mori.

NMR spectroscopic changes as a function of pH in solutions of the pheromone-binding protein of Bombyx mori (BmPBP) show that BmPBP undergoes a conformational transition between pH 4.9 and 6.0. At pH below 4.9 there is a single "acid form" (A), and a homogeneous "basic form" (B) exists at pH above 6.0. Between pH 5 and 6, BmPBP exists as a mixture of A and B in slow exchange on the NMR chemical shift time scale, with the transition midpoint at pH 5.4. The form B has a well-dispersed NMR spectrum, indicating that it represents a more structured, "closed" conformation than form A, which has a significantly narrower chemical shift dispersion. Conformational transitions of the kind observed here may explain heterogeneity reported for a variety of odorant-binding proteins, and it will be of interest to further investigate possible correlations with pH-dependent regulation of ligand binding and release in the biological function of this class of proteins.

Animals↗
PubMed1999

Conformational changes of the BS2 operator DNA upon complex formation with the Antennapedia homeodomain studied by NMR with 13C/15N-labeled DNA.

The NMR structures have been determined for a 13C/15N doubly labeled 14 base-pair DNA duplex comprising the BS2 operator sequence both free in solution and in the complex with the Antennapedia homeodomain. The impact of the DNA labeling is assessed from comparison with a previous structure of the same complex that was determined using isotope labeling only for the protein. Differences between the two structure determinations are nearly completely limited to the DNA, which retains the global B -conformation of the free DNA also in the complex. Local protein-induced conformational changes are a narrowing of the minor groove due to the interaction with the N-terminal arm of the homeodomain, and changes of the sugar puckers of the deoxyriboses G5 and C6, which are apparently induced by van der Waals interactions with Tyr25, and with Gln50 and Arg53, respectively. The high conservation of these amino acid residues in homeodomains suggests that protein-induced shifts in some sugar puckers contribute to the affinity of homeodomains to their cognate DNA. The data obtained here with the Antennapedia homeodomain-DNA complex clearly show that nucleic acid isotope-labeling can support detailed conformational characterization of DNA in complexes with proteins, which will be indispensable for structure determinations of complexes containing globally distorted DNA conformations.

Antennapedia Homeodomain Protein↗
PubMed1999

The 3D NOESY-[(1)H,(15)N,(1)H]-ZQ-TROSY NMR experiment with diagonal peak suppression.

In our 3D NOESY-[(1)H,(15)N,(1)H]-ZQ-TROSY experiment, the TROSY principle (transverse relaxation-optimized spectroscopy) is used in three-dimensional (3D) (15)N-resolved nuclear Overhauser enhancement spectroscopy (NOESY), which enables resonance assignments by sequential nuclear Overhauser effects and the collection of structural constraints in large (15)N- or (2)H,(15)N-labeled proteins. Our experiment affords optimization of the transverse relaxation in all three frequency dimensions, provides suppression of the strong diagonal autorelaxation peaks, which otherwise tend to interfere with the analysis of nearby informative crosspeaks, and yields improved resolution for the entire spectrum when compared with conventional 3D (15)N-resolved-[(1)H,(1)H]-NOESY, because of the narrower lineshapes along both proton dimensions. The key element of this experiment is an approach for correlating the (15)N and (1)H chemical shifts with two-dimensional ZQ-[(15)N,(1)H]-TROSY, where zero-quantum (ZQ) coherence is generated and the remote cross-correlation between the (1)H and (15)N chemical shift anisotropy interactions is used to reduce transverse relaxation during (15)N evolution. Practical applications are illustrated with spectra of a protein with a molecular mass of 110,000 Da.

Magnetic Resonance Spectroscopy↗
PubMed1999

NMR structure of the sea urchin (Strongylocentrotus purpuratus) metallothionein MTA.

The three-dimensional structure of [(113)Cd7]-metallothionein-A (MTA) of the sea urchin Strongylocentrotus purpuratus was determined by homonuclear(1)H NMR experiments and heteronuclear [(1)H, (113)Cd]-correlation spectroscopy. MTA is composed of two globular domains, an N-terminal four-metal domain of the amino acid residues 1 to 36 and a Cd4Cys11cluster, and a C-terminal three-metal domain including the amino acid residues 37 to 65 and a Cd3Cys9cluster. The structure resembles the known mammalian and crustacean metallothioneins, but has a significantly different connectivity pattern of the Cys-metal co-ordination bonds and concomitantly contains novel local folds of some polypeptide backbone segments. These differences can be related to variations of the Cys sequence positions and thus emphasize the special role of the cysteine residues in defining the structure of metallothioneins, both on the level of the domain architecture and the topology of the metal-thiolate clusters.

Amino Acid Sequence↗
PubMed1999

NMR structure and metal interactions of the CopZ copper chaperone.

A recently discovered family of proteins that function as copper chaperones route copper to proteins that either require it for their function or are involved in its transport. In Enterococcus hirae the copper chaperone function is performed by the 8-kDa protein CopZ. This paper describes the NMR structure of apo-CopZ, obtained using uniformly (15)N-labeled CopZ overexpressed in Escherichia coli and NMR studies of the impact of Cu(I) binding on the CopZ structure. The protein has a betaalphabetabetaalphabeta fold, where the four beta-strands form an antiparallel twisted beta-sheet, and the two helices are located on the same side of the beta-sheet. A sequence motif GMXCXXC in the loop between the first beta-strand and the first alpha-helix contains the primary ligands, which bind copper(I). Binding of copper(I) caused major structural changes in this molecular region, as manifested by the fact that most NMR signals of the loop and the N-terminal part of the first helix were broadened beyond detection. This effect was strictly localized, because the remainder of the apo-CopZ structure was maintained after addition of Cu(I). NMR relaxation data showed a decreased correlation time of overall molecular tumbling for Cu(I)-CopZ when compared with apo-CopZ, indicating aggregation of Cu(I)-CopZ. The structure of CopZ is the first three-dimensional structure of a cupro-protein for which the metal ion is an exchangeable substrate rather than an integral part of the structure. Implications of the present structural work for the in vivo function of CopZ are discussed, whereby it is of special interest that the distribution of charged residues on the CopZ surface is highly uneven and suggests preferred recognition sites for other proteins that might be involved in copper transfer.

Amino Acid Sequence↗
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↗