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Hartmut Oschkinat

Publications and source records attributed to Hartmut Oschkinat.

17 recordsLinked to original sources
PubMed2003

Design of N-substituted peptomer ligands for EVH1 domains.

Ena/VASP proteins are implicated in cytoskeletal reorganization during actin-dependent motility processes. Recruitment to subcellular sites of actin polymerization is mediated by the highly conserved N-terminal EVH1 domain, which interacts with target proteins containing proline-rich motifs. The VASP EVH1 domain specifically binds peptides with the consensus motif FPPPP present in all its binding partners, including the Listerial ActA protein. Previous studies have shown that the Phe and first and final Pro residues are highly conserved and cannot be substituted with any other natural amino acid without significant loss of binding affinity. We have incorporated peptoid building blocks (sarcosine derived, non-natural amino acids) into the peptide SFEFPPPPTEDEL from the Listerial ActA protein and were able to substitute the most highly conserved residues of this motif while maintaining binding to the VASP EVH1 domain with affinities in the range of 45-180 microm. We then used NMR chemical shift perturbations to locate specific domain residues involved in particular interactions. These studies may open up the way for designing selective modulators of VASP function for biological studies and for the development of novel therapeutics for diseases involving pathologically altered cell adhesion or cell motility.

Actins↗
PubMed2003

1H detection in MAS solid-state NMR spectroscopy of biomacromolecules employing pulsed field gradients for residual solvent suppression.

In this communication, we demonstrate the feasibility of 1H detection in MAS solid-state NMR for a microcrystalline, uniformly 2H,15N-labeled sample of a SH3 domain of chicken alpha-spectrin, using pulsed field gradients for suppression of water magnetization. Today, B0 gradients are employed routinely in solution-state NMR for coherence order selection and solvent suppression. We suggest to use gradients to purge water magnetization which cannot be suppressed using conventional water suppression schemes. The achievable gain in sensitivity for 1H detection is in the order of 5 compared to the 15N detected version of the experiment (at a MAS rotation frequency of 13.5 kHz). We expect that this labeling concept which achieves high sensitivity due to 1H detection, in combination with the possibility to measure long range 1H-1H distances as we have shown previously, to be a useful tool for the determination of protein structures in the solid state.

Animals↗
PubMed2003

Measurement of multiple psi torsion angles in uniformly 13C,15N-labeled alpha-spectrin SH3 domain using 3D 15N-13C-13C-15N MAS dipolar-chemical shift correlation spectroscopy.

We demonstrate the simultaneous measurement of several backbone torsion angles psi in the uniformly (13)C,(15)N-labeled alpha-Spectrin SH3 domain using two different 3D 15N-13C-13C-15N dipolar-chemical shift magic-angle spinning (MAS) NMR experiments. The first NCCN experiment utilizes double quantum (DQ) spectroscopy combined with the INADEQUATE type 13C-13C chemical shift correlation. The decay of the DQ coherences formed between 13C'(i) and 13C(alphai) spin pairs is determined by the "correlated" dipolar field due to 15N(i)-13C(alphai) and 13C'(i)-15N(i+1) dipolar couplings and is particularly sensitive to variations of the torsion angle in the regime |psi| > 140 degrees. However, the ability of this experiment to constrain multiple psi-torsion angles is limited by the resolution of the 13C(alpha)-(13)CO correlation spectrum. This problem is partially addressed in the second approach described here, which is an NCOCA NCCN experiment. In this case the resolution is enhanced by the superior spectral dispersion of the 15N resonances present in the 15N(i+1)-13C(alphai) part of the NCOCA chemical shift correlation spectrum. For the case of the 62-residue alpha-spectrin SH3 domain, we determined 13 psi angle constraints with the INADEQUATE NCCN experiment and 22 psi constraints were measured in the NCOCA NCCN experiment.

Carbon Isotopes↗
PubMed2003

The ScPex13p SH3 domain exposes two distinct binding sites for Pex5p and Pex14p.

Pex13p is an essential component of the peroxisomal protein import machinery and interacts via its C-terminal SH3 domain with the type II SH3-ligand Pex14p and the non-PXXP protein Pex5p. We report the solution structure of the SH3 domain of Pex13p from Saccharomyces cerevisiae and the identification of a novel-binding pocket, which binds a non-PXXP-peptide representing the binding site of Pex5p. Chemical shift assays revealed the binding sites for Pex5p and Pex14p ligand peptides to be distinct and spatially separated. Competition assays demonstrated that the two ligand peptides can bind simultaneously to the SH3 domain.

Amino Acid Motifs↗
PubMed2003

Characterization of (1)H-(1)H distances in a uniformly (2)H,(15)N-labeled SH3 domain by MAS solid-state NMR spectroscopy (section sign).

In this communication, we demonstrate the feasibility of obtaining long-range (1)H-(1)H distance information by MAS solid-state NMR for a microcrystalline, uniformly (2)H,(15)N-labeled sample of a SH3 domain of chicken alpha-spectrin. The experiments yield NOESY-type spectra and rely on the favorable dispersion of the (15)N chemical shifts of the protein backbone. Perdeuteration of nonexchangeable sites is employed to simplify proton spin systems and to obtain multiple structural information. Two mixing schemes, (1)H-(1)H double quantum filtered Post-C7 and (1)H spin diffusion, are implemented to obtain quantitative (1)H-(1)H distance information. Post-C7 and spin diffusion cross-peak buildup rates are discussed for initial-rate fitting and in the framework of n = 0 rotational resonance (rotor driven spin diffusion), respectively. Different deuteration schemes were tested to find conditions where short-range (1)H-(1)H interactions are truncated (e.g., between H(N) and H(alpha)), but long-range interactions are retained (e.g., between H(N) and H(N)).

Animals↗
PubMed2003

Assignment of amide proton signals by combined evaluation of HN, NN and HNCA MAS-NMR correlation spectra.

In this paper, we present a strategy for the (1)H(N) resonance assignment in solid-state magic-angle spinning (MAS) NMR, using the alpha-spectrin SH3 domain as an example. A novel 3D triple resonance experiment is presented that yields intraresidue H(N)-N-C(alpha) correlations, which was essential for the proton assignment. For the observable residues, 52 out of the 54 amide proton resonances were assigned from 2D ((1)H-(15)N) and 3D ((1)H-(15)N-(13)C) heteronuclear correlation spectra. It is demonstrated that proton-driven spin diffusion (PDSD) experiments recorded with long mixing times (4 s) are helpful for confirming the assignment of the protein backbone (15)N resonances and as an aid in the amide proton assignment.

Amides↗
PubMed2003

WW domain sequence activity relationships identified using ligand recognition propensities of 42 WW domains.

WW domains mediate protein-protein interactions in a number of different cellular functions by recognizing proline-containing peptide sequences. We determined peptide recognition propensities for 42 WW domains using NMR spectroscopy and peptide library screens. As potential ligands, we studied both model peptides and peptides based on naturally occurring sequences, including phosphorylated residues. Thirty-two WW domains were classified into six groups according to detected ligand recognition preferences for binding the motifs PPx(Y/poY), (p/phi)P(p,g)PPpR, (p/phi)PPRgpPp, PPLPp, (p/xi)PPPPP, and (poS/poT)P (motifs according to modified Seefeld Convention 2001). In addition to these distinct binding motifs, group-specific WW domain consensus sequences were identified. For PPxY-recognizing domains, phospho-tyrosine binding was also observed. Based on the sequences of the PPx(Y/poY)-specific group, a profile hidden Markov model was calculated and used to predict PPx(Y/poY)-recognition activity for WW domains, which were not assayed. PPx(Y/poY)-binding was found to be a common property of NEDD4-like ubiquitin ligases.

Amino Acid Motifs↗
PubMed2002

Structure of a protein determined by solid-state magic-angle-spinning NMR spectroscopy.

The determination of a representative set of protein structures is a chief aim in structural genomics. Solid-state NMR may have a crucial role in structural investigations of those proteins that do not easily form crystals or are not accessible to solution NMR, such as amyloid systems or membrane proteins. Here we present a protein structure determined by solid-state magic-angle-spinning (MAS) NMR. Almost complete (13)C and (15)N resonance assignments for a micro-crystalline preparation of the alpha-spectrin Src-homology 3 (SH3) domain formed the basis for the extraction of a set of distance restraints. These restraints were derived from proton-driven spin diffusion (PDSD) spectra of biosynthetically site-directed, labelled samples obtained from bacteria grown using [1,3-(13)C]glycerol or [2-(13)C]glycerol as carbon sources. This allowed the observation of long-range distance correlations up to approximately 7 A. The calculated global fold of the alpha-spectrin SH3 domain is based on 286 inter-residue (13)C-(13)C and six (15)N-(15)N restraints, all self-consistently obtained by solid-state MAS NMR. This MAS NMR procedure should be widely applicable to small membrane proteins that can be expressed in bacteria.

Animals↗
PubMed2002

Relaxation, equilibrium oligomerization, and molecular symmetry of the VASP (336-380) EVH2 tetramer.

An investigation of the structural and dynamic properties of the C-terminal fragment of the human protein VASP (VASP 336-380) has been performed. Full length VASP has been shown to be tetrameric in solution, and the C-terminal 45 residues of the protein have been suggested to be responsible for the oligomerization. We have expressed and purified a C-terminal fragment of the human VASP protein from residue 336-380. It was found to form a stable domain in its own right. The fragment was shown by CD spectroscopy to form a helical structure, stable under a wide range of temperature and pH conditions. A (15)N-HSQC-experiment exhibits only one set of peaks, suggesting a high degree of symmetry for a putative oligomer. Measurements of the rotational correlation time tau(C) of the molecule and analytical ultracentrifugation data show VASP (336-380) to be entirely tetrameric in solution. The secondary structure was confirmed from a (15)N-NOESY-HSQC experiment and is completely alpha-helical. We conclude that VASP (336-380) forms a tetramer in solution via a coiled coil arrangement and is solely responsible for tetramerization of full-length VASP.

Amino Acid Sequence↗
PubMed2002

Biosynthesis of riboflavin in archaea studies on the mechanism of 3,4-dihydroxy-2-butanone-4-phosphate synthase of Methanococcus jannaschii.

The hypothetical protein predicted by the open reading frame MJ0055 of Methanococcus jannaschii was expressed in a recombinant Escherichia coli strain under the control of a synthetic gene optimized for translation in an eubacterial host. The recombinant protein catalyzes the formation of the riboflavin precursor 3,4-dihydroxy-2-butanone 4-phosphate from ribulose 5-phosphate at a rate of 174 nmol mg(-1) min(-1) at 37 degrees C. The homodimeric 51.6-kDa protein requires divalent metal ions, preferentially magnesium, for activity. The reaction involves an intramolecular skeletal rearrangement as shown by (13)C NMR spectroscopy using [U-(13)C(5)]ribulose 5-phosphate as substrate. A cluster of charged amino acid residues comprising arginine 25, glutamates 26 and 28, and aspartates 21 and 30 is essential for catalytic activity. Histidine 164 and glutamate 185 were also shown to be essential for catalytic activity.

Amino Acid Sequence↗
PubMed2002

The structures of the active center in dark-adapted bacteriorhodopsin by solution-state NMR spectroscopy.

The two forms of bacteriorhodopsin present in the dark-adapted state, containing either all-trans or 13-cis,15-syn retinal, were examined by using solution state NMR, and their structures were determined. Comparison of the all-trans and the 13-cis,15-syn forms shows a shift in position of about 0.25 A within the pocket of the protein. Comparing this to the 13-cis,15-anti chromophore of the catalytic cycle M-intermediate structure, the 13-cis,15-syn form demonstrates a less pronounced up-tilt of the retinal C12[bond]C14 region, while leaving W182 and T178 essentially unchanged. The N[bond]H dipole of the Schiff base orients toward the extracellular side in both forms, however, it reorients toward the intracellular side in the 13-cis,15-anti configuration to form the catalytic M-intermediate. Thus, the change of the N[bond]H dipole is considered primarily responsible for energy storage, conformation changes of the protein, and the deprotonation of the Schiff base. The structural similarity of the all-trans and 13-cis,15-syn forms is taken as strong evidence for the ion dipole dragging model by which proton (hydroxide ion) translocation follows the change of the dipole.

Amino Acid Sequence↗
PubMed2002

EVH1 domains: structure, function and interactions.

Drosophila enabled/vasodilator-stimulated phosphoprotein homology 1 (EVH1) domains are 115 residue protein-protein interaction modules which provide essential links for their host proteins to various signal transduction pathways. Many EVH1-containing proteins are associated closely with actin-based structures and are involved in re-organization of the actin cytoskeleton. EVH1 domains are also present in proteins enriched in neuronal tissue, thus implicating them as potential mediators of synaptic plasticity, linking them to memory formation and learning. Like Src homology 3, WW and GYF domains and profilin, EVH1 domains recognize and bind specific proline-rich sequences (PRSs). The binding is of low affinity, but tightly regulated by the high specificity encoded into residues in the protein:peptide interface. In general, a small (3-6 residue) 'core' PRS in the target protein binds a 'recognition pocket' on the domain surface. Further affinity- and specificity-increasing interactions are then formed between additional domain epitopes and peptide 'core-flanking' residues. The three-dimensional structures of EVH1:peptide complexes now reveal, in great detail, some of the most important features of these interactions and allow us to better understand the origins of specificity, ligand orientation and sequence degeneracy of target peptides, in low affinity signalling complexes.

Amino Acid Sequence↗
PubMed2002

A 3-D structural model of solid self-assembled chlorophyll a/H(2)O from multispin labeling and MAS NMR 2-D dipolar correlation spectroscopy in high magnetic field.

Magic angle spinning (MAS) NMR with Lee-Goldburg cross-polarization (LG-CP) is used to promote long-range heteronuclear transfer of magnetization and to constrain a structural model for uniformly labeled chlorophyll a/H(2)O. An effective maximum transfer range d(max) can be determined experimentally from the detection of a gradually decreasing series of intramolecular correlations with the (13)C along the molecular skeleton. To probe intermolecular contacts, d(max) can be set to approximately 4.2 A by choosing an LG-CP contact time of 2 ms. Long-range (1)H-(13)C correlations are used in conjunction with carbon and proton aggregation shifts to establish the stacking of the chlorophyll a (Chl a) molecules. First, high-field (14.1 T) 2-D MAS NMR homonuclear ((13)C-(13)C) dipolar correlation spectra provide a complete assignment of the carbon chemical shifts. Second, proton chemical shifts are obtained from (1)H-(13)C heteronuclear dipolar correlation spectroscopy in high magnetic field. The shift constraints and long-range (1)H-(13)C intermolecular correlations reveal a 2-D stacking homologous to the molecular arrangement in crystalline solid ethyl-chlorophyllide a. A doubling of a small subset of the carbon resonances, in the 7-methyl region of the molecule, provides evidence for two marginally different well-defined molecular environments. Evidence is found for the presence of neutral structural water molecules forming a hydrogen-bonded network to stabilize Chl a sheets. In line with the microcrystalline order observed for the rings, the long T(1)'s, and absence of conformational shifts for the (13)C in the phytyl tails, it is proposed that the Chl a form a rigid 3-D space-filling structure. Probably the only way this can be realized with the sheets is by forming bilayers with interpenetration of elongated tails. Such a 3-D space-filling organization of the aggregated Chl a from MAS NMR would match existing models inferred from electron microscopy and low-resolution X-ray powder diffraction, while a micellar model based on neutron diffraction and antiparallel stacking observed in solution can be discarded.

Carbon Isotopes↗
PubMed2002

2D(13)C-(13)C MAS NMR correlation spectroscopy with mixing by true (1)H spin diffusion reveals long-range intermolecular distance restraints in ultra high magnetic field.

An improved 2D (13)C-(13)C CP(3) MAS NMR correlation experiment with mixing by true (1)H spin diffusion is presented. With CP(3), correlations can be detected over a much longer range than with direct (1)H-(13)C or (13)C-(13)C dipolar recoupling. The experiment employs a (1)H spin diffusion mixing period tau(m) sandwiched between two cross-polarization periods. An optimized CP(3) sequence for measuring polarization transfer on a length scale between 0.3 and 1.0 nm using short mixing times of 0.1 ms < tau(m) < 1 ms is presented. For such a short tau(m), cross talk from residual transverse magnetization of the donating nuclear species after a CP can be suppressed by extended phase cycling. The utility of the experiment for genuine structure determination is demonstrated using a self-aggregated Chl a/H(2)O sample. The number of intramolecular cross-peaks increases for longer mixing times and this obscures the intermolecular transfer events. Hence, the experiment will be useful for short mixing times only. For a short tau(m) = 0.1 ms, intermolecular correlations are detected between the ends of phytyl tails and ring carbons of neighboring Chl a molecules in the aggregate. In this way the model for the structure, with stacks of Chl a that are arranged back to back with interdigitating phytyl chains stretched between two bilayers, is validated.

Journal Article↗
PubMed2002

A software tool for the prediction of Xaa-Pro peptide bond conformations in proteins based on 13C chemical shift statistics.

The chemical shift difference (delta[13C(beta)]-delta[13C(gamma)]) is a reference-independent indicator of the Xaa-Pro peptide bond conformation. Based on a statistical analysis of the 13C chemical shifts of 1,033 prolines from 304 proteins deposited in the BioMagRes database, a software tool was created to predict the probabilities for cis or trans conformations of Xaa-Pro peptide bonds. Using this approach, the conformation at a given Xaa-Pro bond can be identified in a simple NOE-independent way immediately after obtaining its NMR resonance assignments. This will allow subsequent structure calculations to be initiated using the correct polypeptide chain conformation.

Amino Acids↗