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Gerhard Wider

Publications and source records attributed to Gerhard Wider.

At least 19 recordsLinked to original sources
PubMed2006

Proton-proton Overhauser NMR spectroscopy with polypeptide chains in large structures.

The use of 1H-1H nuclear Overhauser effects (NOE) for structural studies of uniformly deuterated polypeptide chains in large structures is investigated by model calculations and NMR experiments. Detailed analysis of the evolution of the magnetization during 1H-1H NOE experiments under slow-motion conditions shows that the maximal 1H-1H NOE transfer is independent of the overall rotational correlation time, even in the presence of chemical exchange with the bulk water, provided that the mixing time is adjusted for the size of the structure studied. 1H-1H NOE buildup measurements were performed for the 472-kDa complex of the 72-kDa cochaperonin GroES with a 400-kDa single-ring variant of the chaperonin GroEL (SR1). These experiments demonstrate that multidimensional NOESY experiments with cross-correlated relaxation-enhanced polarization transfer and transverse relaxation-optimized spectroscopy elements can be applied to structures of molecular masses up to several hundred kilodaltabs, which opens new possibilities for studying functional interactions in large maromolecular assemblies in solution.

Chaperonin 10↗
PubMed2006

Automated resonance assignment of proteins: 6D APSY-NMR.

The 6-dimensional (6D) APSY-seq-HNCOCANH NMR experiment correlates two sequentially neighboring amide moieties in proteins via the C' and Calpha nuclei, with efficient suppression of the back transfer from Calpha to the originating amide moiety. The automatic analysis of two-dimensional (2D) projections of this 6D experiment with the use of GAPRO (Hiller et al., 2005) provides a high-precision 6D peak list, which permits automated sequential assignments of proteins with the assignment software GARANT (Bartels et al., 1997). The procedure was applied to two proteins, the 63-residue 434-repressor(1-63) and the 115-residue TM1290. For both proteins, complete sequential assignments for all NMR-observable backbone resonances were obtained, and the polypeptide segments thus identified could be unambiguously located in the amino acid sequence. These results demonstrate that APSY-NMR spectroscopy in combination with a suitable assignment algorithm can provide fully automated sequence-specific backbone assignments of small proteins.

Algorithms↗
PubMed2006

Measuring protein concentrations by NMR spectroscopy.

In applications of NMR to biological macromolecules in solution, the concentration of the NMR sample is an important parameter describing the sample and providing information for the selection and planning of experiments. Although concentrations can be measured directly by NMR spectroscopy, other methods are usually preferred to measure the concentration of macromolecules in NMR samples. The reasons are the difficulties in the correlation of the sample of interest with the signal intensity representing a known concentration. This correlation is usually obtained by adding to the sample a reference compound with known concentration and comparing the integral over resolved resonance lines of the molecules with known and unknown concentrations. For solutions of biological macromolecules it is very difficult to find a compound that does not interact with the macromolecules and has a resonance outside their spectral range. We introduce PULCON which is a method that correlates the absolute intensities of two spectra measured in different solution conditions. PULCON is easy to implement and apply on all NMR spectrometers; it does not need any special hardware or software. PULCON is very robust and at the same time delivers accurate concentrations of samples in the NMR tube. We demonstrate that PULCON has the potential to replace UV spectroscopy for concentration measurements of NMR samples.

Egg Proteins↗
PubMed2006

NMR-derived dynamic aspects of N-type inactivation of a Kv channel suggest a transient interaction with the T1 domain.

Some eukaryotic voltage-gated K+ (Kv) channels contain an N-terminal inactivation peptide (IP), which mediates a fast inactivation process that limits channel function during membrane depolarization and thus shapes the action potential. We obtained sequence-specific nuclear magnetic resonance (NMR) assignments for the polypeptide backbone of a tetrameric N-terminal fragment (amino acids 1-181) of the Aplysia Kv1.1 channel. Additional NMR measurements show that the tetramerization domain 1 (T1) has the same globular structure in solution as previously determined by crystallography and that the IP (residues 1-20) and the linker (residues 21-65) are in a flexibly disordered, predominantly extended conformation. A potential contact site between the T1 domain and the flexible tail (residues 1-65) has been identified on the basis of chemical-shift changes of individual T1 domain amino acids, which map to the T1 surface near the interface between adjacent subunits. Paramagnetic perturbation experiments further indicate that, in the ensemble of solution conformers, there is at least a small population of species with the IP localized in close proximity to the proposed interacting residues of the T1 tetramer. Electrophysiological measurements show that all three mutations in this pocket that we tested slow the rate of inactivation and speed up recovery, as predicted from the preinactivation site model. These results suggest that specific, short-lived transient interactions between the T1 domain and the IP or the linker segment may play a role in defining the regulatory kinetics of fast channel inactivation.

Amino Acid Sequence↗
PubMed2006

Concentration measurements by PULCON using X-filtered or 2D NMR spectra.

Sample concentrations can be measured by nuclear magnetic resonance (NMR) spectroscopy without an internal reference compound using pulse length based concentration determination (PULCON) with 1D NMR spectra. PULCON delivers most accurate results if the spectrum of the sample of interest contains a resolved resonance; but can also be applied to spectral regions with overlapping resonances. If the exact number of lines contributing to the overlapping spectral region is not known, a corresponding error makes the result less precise. The uncertainty about the number of contributing resonances can be reduced with experiments that discriminate different classes of resonances by filtering techniques or by extending PULCON to two-dimensional NMR spectra. We demonstrate the application of PULCON with a 1D 15N-filtered experiment where aromatic resonances of a protein can be observed without interference from 15N-bound protons. Further, we extend PULCON to 2D NMR spectra that permits to determine the exact number of resonances. This extension can readily be applied with sample that contain different solutes.

Amino Acids, Aromatic↗
PubMed2005

Ubiquitin-binding domains in Y-family polymerases regulate translesion synthesis.

Translesion synthesis (TLS) is the major pathway by which mammalian cells replicate across DNA lesions. Upon DNA damage, ubiquitination of proliferating cell nuclear antigen (PCNA) induces bypass of the lesion by directing the replication machinery into the TLS pathway. Yet, how this modification is recognized and interpreted in the cell remains unclear. Here we describe the identification of two ubiquitin (Ub)-binding domains (UBM and UBZ), which are evolutionarily conserved in all Y-family TLS polymerases (pols). These domains are required for binding of poleta and poliota to ubiquitin, their accumulation in replication factories, and their interaction with monoubiquitinated PCNA. Moreover, the UBZ domain of poleta is essential to efficiently restore a normal response to ultraviolet irradiation in xeroderma pigmentosum variant (XP-V) fibroblasts. Our results indicate that Ub-binding domains of Y-family polymerases play crucial regulatory roles in TLS.

Amino Acid Motifs↗
PubMed2005

Effective rotational correlation times of proteins from NMR relaxation interference.

Knowledge of the effective rotational correlation times, tauc, for the modulation of anisotropic spin-spin interactions in macromolecules subject to Brownian motion in solution is of key interest for the practice of NMR spectroscopy in structural biology. The value of tauc enables an estimate of the NMR spin relaxation rates, and indicates possible aggregation of the macromolecular species. This paper reports a novel NMR pulse scheme, [15N,1H]-TRACT, which is based on transverse relaxation-optimized spectroscopy and permits to determine tauc for 15N-1H bonds without interference from dipole-dipole coupling of the amide proton with remote protons. [15N,1H]-TRACT is highly efficient since only a series of one-dimensional NMR spectra need to be recorded. Its use is suggested for a quick estimate of the rotational correlation time, to monitor sample quality and to determine optimal parameters for complex multidimensional NMR experiments. Practical applications are illustrated with the 110 kDa 7,8-dihydroneopterin aldolase from Staphylococcus aureus, the uniformly 15N-labeled Escherichia coli outer membrane protein X (OmpX) in 60 kDa mixed OmpX/DHPC micelles with approximately 90 molecules of unlabeled 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), and the 16 kDa pheromone-binding protein from Bombyx mori, which cover a wide range of correlation times.

Aldehyde-Lyases↗
PubMed2005

Direct NMR observation of a substrate protein bound to the chaperonin GroEL.

The reaction cycle and the major structural states of the molecular chaperone GroEL and its cochaperone, GroES, are well characterized. In contrast, very little is known about the nonnative states of the substrate polypeptide acted on by the chaperonin machinery. In this study, we investigated the substrate protein human dihydrofolate reductase (hDHFR) while bound to GroEL or to a single-ring analog, SR1, by NMR spectroscopy in solution under conditions where hDHFR was efficiently recovered as a folded, enzymatically active protein from the stable complexes upon addition of ATP and GroES. By using the NMR techniques of transverse relaxation-optimized spectroscopy (TROSY), cross-correlated relaxation-induced polarization transfer (CRIPT), and cross-correlated relaxation-enhanced polarization transfer (CRINEPT), bound hDHFR could be observed directly. Measurements of the buildup of hDHFR NMR signals by different magnetization transfer mechanisms were used to characterize the dynamic properties of the NMR-observable parts of the bound substrate. The NMR data suggest that the bound state includes random coil conformations devoid of stable native-like tertiary contacts and that the bound hDHFR might best be described as a dynamic ensemble of randomly structured conformers.

Chaperonin 10↗
PubMed2005

Automated projection spectroscopy (APSY).

This work presents the automated projection spectroscopy (APSY) method for the recording of discrete sets of j projections from N-dimensional (N > or = 3) NMR experiments at operator-selected projection angles and automatic identification of the correlation cross peaks. The result from APSY is the fully automated generation of the complete or nearly complete peak list for the N-dimensional NMR spectrum from a geometric analysis of the j experimentally recorded, low-dimensional projections. In the present implementation of APSY, two-dimensional projections of the N-dimensional spectrum are recorded by using techniques developed for projection-reconstruction spectroscopy [Kupce,E.& Freeman, R. (2004) J. Am. Chem. Soc. 126, 6429-6440]. All projections are peak-picked with the available automated routine atnos. The previously undescribed algorithm GAPRO (geometric analysis of projections) uses vector algebra to identify subgroups of peaks in different projections that arise from the same resonance in the N-dimensional spectrum, and from these subgroups it calculates the peak positions in the N-dimensional frequency space. Unambiguous identification thus can be achieved for all cross peaks that are not overlapped with other peaks in at least one of the N dimensions. Because of the correlation between the positions of corresponding peaks in multiple projections, uncorrelated noise is efficiently suppressed, so that APSY should be quite widely applicable for correlation spectra of biological macromolecules, which have intrinsically low peak density in the N-dimensional spectral space.

Journal Article↗
PubMed2005

Managing the solvent water polarization to obtain improved NMR spectra of large molecular structures.

In large molecular structures, the magnetization of all hydrogen atoms in the solute is strongly coupled to the water magnetization through chemical exchange between solvent water and labile protons of macromolecular components, and through dipole-dipole interactions and the associated "spin diffusion" due to slow molecular tumbling. In NMR experiments with such systems, the extent of the water polarization is thus of utmost importance. This paper presents a formalism that describes the propagation of the water polarization during the course of different NMR experiments, and then compares the results of model calculations for optimized water polarization with experimental data. It thus demonstrates that NMR spectra of large molecular structures can be improved with the use of paramagnetic spin relaxation agents which selectively enhance the relaxation of water protons, so that a substantial gain in signal-to-noise can be achieved. The presently proposed use of a relaxation agent can also replace the water flip-back pulses when working with structures larger than about 30 kDa. This may be a valid alternative in situations where flip-back pulses are difficult to introduce into the overall experimental scheme, or where they would interfere with other requirements of the NMR experiment.

Chaperonin 60↗
PubMed2005

NMR techniques used with very large biological macromolecules in solution.

Methods for the characterization of very large biological macromolecules by NMR in solution are presented. For studies of molecular structures with molecular weights beyond 100,000 Da transverse relaxation in common multidimensional NMR experiments becomes a limiting factor. Novel techniques optimize transverse relaxation based on cross-correlated relaxation between dipole-dipole interactions and chemical shift anisotropy (CSA), and include transverse relaxation-optimized spectroscopy (TROSY), cross-correlated relaxation-enhanced polarization transfer (CRINEPT), and cross-correlated relaxation-induced polarization transfer (CRIPT). In combination with various biochemical isotope-labeling techniques these experimental schemes make possible studies of biological macromolecules with molecular masses of up to 1,000,000 Da by NMR in solution. The physical basis and the implementation of the experiments are discussed.

Macromolecular Substances↗
PubMed2004

NMR structures of salt-refolded forms of the 434-repressor DNA-binding domain in 6 M urea.

The N-terminal 63-residue fragment of the phage 434-repressor, 434(1-63), has a well-defined globular fold in H(2)O solution, and is unfolded in 6 M urea at pH 7.5. In this study, 434(1-63) has been refolded by adding either 1.7 M NaCl or 0.47 M NaTFA to the solution in 6 M urea, and the NMR structures of both refolded forms have been determined. The two refolded forms have similar free energies of unfolding and are approximately 16 kJ/mol less stable than the protein in H(2)O solution. 434(1-63) refolded with NaCl exhibits NMR chemical shifts very similar to and a three-dimensional structure nearly identical to those of 434(1-63) in H(2)O solution. The protein refolded with NaTFA also has a similar global fold, but it shows local differences near Phe44, of which two different orientations of the aromatic ring are compatible with the experimental data. This local conformational polymorphism attracted our interest because hydrophobic contacts between two subdomains of residues 1-36 and 45-63 are mediated by the Phe44 side chain. Anion binding experiments suggest that this polymorphism is caused by binding of TFA(-) anions to a cluster of positively charged Arg and Lys residues located in the loop connecting the two subdomains, with apparent binding constants for TFA(-) (K(app)) on the order of 30 mM(-1).

Anions↗
PubMed2004

Membrane protein-lipid interactions in mixed micelles studied by NMR spectroscopy with the use of paramagnetic reagents.

For solution NMR studies of the structure and function of membrane proteins, these macromolecules have to be reconstituted and solubilized in detergent micelles. Detailed characterization of the mixed detergent/protein micelles is then of key importance to validate the results from such studies, and to evaluate how faithfully the natural environment of the protein in the biological membrane is mimicked by the micelle. In this paper, a selection of paramagnetic probes with different physicochemical properties are used to characterize the 60 kDa mixed micelles consisting of about 90 molecules of the detergent dihexanoylphosphatidylcholine (DHPC) and one molecule of the Escherichia coli outer-membrane protein X (OmpX), which had previously been extensively studied by solution NMR techniques. The observation of highly selective relaxation effects on the NMR spectra of OmpX and DHPC from a water-soluble relaxation agent and from nitroxide spin labels attached to lipophilic molecules, confirmed data obtained previously with more complex NMR studies of the diamagnetic OmpX/DHPC system, and yielded additional novel insights into the protein-detergent interactions in the mixed micelles. The application of paramagnetic probes to the well-characterized OmpX/DHPC system indicates that such probes should be widely applicable as an efficient support of NMR studies of the topology of mixed membrane protein-detergent micelles.

Bacterial Outer Membrane Proteins↗
PubMed2004

NMR structure of the integral membrane protein OmpX.

The structure of the integral membrane protein OmpX from Escherichia coli reconstituted in 60 kDa DHPC micelles (OmpX/DHPC) was calculated from 526 NOE upper limit distance constraints. The structure determination was based on complete sequence-specific assignments for the amide protons and the Val, Leu, and Ile(delta1) methyl groups in OmpX, which were selectively protonated on a perdeuterated background. The solution structure of OmpX in the DHPC micelles consists of a well-defined, eight-stranded antiparallel beta-barrel, with successive pairs of beta-strands connected by mobile loops. Several long-range NOEs observed outside of the transmembrane barrel characterize an extension of a four-stranded beta-sheet beyond the height of the barrel. This protruding beta-sheet is believed to be involved in intermolecular interactions responsible for the biological functions of OmpX. The present approach for de novo structure determination should be quite widely applicable to membrane proteins reconstituted in mixed micelles with overall molecular masses up to about 100 kDa, and may also provide a platform for additional functional studies.

Amino Acid Sequence↗
PubMed2003

TROSY in NMR studies of the structure and function of large biological macromolecules.

Transverse relaxation-optimized spectroscopy (TROSY), in combination with various isotope-labeling techniques, has opened avenues to study biomolecules with molecular masses of up to 1000000Da by solution NMR. Important recent applications of TROSY include the structure determination of membrane proteins in detergent micelles, structural and functional studies of large proteins in both monomeric form and macromolecular complexes, and investigations of intermolecular interactions in large complexes. TROSY improves the measurement of residual dipolar couplings and the detection of scalar couplings across hydrogen bonds - techniques that promise to further enhance the determination of solution structures of large proteins and oligonucleotides.

Binding Sites↗
PubMed2003

NMR structure of the apoptosis- and inflammation-related NALP1 pyrin domain.

Signaling in apoptosis and inflammation is often mediated by proteins of the death domain superfamily in the Fas/FADD/Caspase-8 or the Apaf-1/Caspase-9 pathways. This superfamily currently comprises the death domain (DD), death effector domain (DED), caspase recruitment domain (CARD), and pyrin domain (PYD) subfamilies. The PYD subfamily is most abundant, but three-dimensional structures are only available for the subfamilies DD, DED, and CARD, which have an antiparallel arrangement of six alpha helices as common fold. This paper presents the NMR structure of PYD of NALP1, a protein that is involved in the innate immune response and is a component of the inflammasome. The structure of NALP1 PYD differs from all other known death domain superfamily structures in that the third alpha helix is replaced by a flexibly disordered loop. This unique feature appears to relate to the molecular basis of familial Mediterranean fever (FMF), a genetic disease caused by single-point mutations.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Stereospecific assignments of the isopropyl methyl groups of the membrane protein OmpX in DHPC micelles.

In NMR studies of large molecular structures, the number of conformational constraints based on NOE measurements is typically limited due to the need for partial deuteration. As a consequence, when using selective protonation of peripheral methyl groups on a perdeuterated background, stereospecific assignments of the diastereotopic methyl groups of Val and Leu can have a particularly large impact on the quality of the NMR structure determination. For example, 3D 15N- and 13C-resolved [1H,1H]-NOESY spectra of the E. Coli membrane protein OmpX in mixed micelles with DHPC, which have an overall molecular weight of about 60 kDa, showed that about 50% of all obtainable NOEs involve the diastereotopic methyl groups of Val and Leu. In this paper, we used biosynthetically-directed fractional 13C labeling of OmpX and [13C,1H]-HSQC spectroscopy to obtain stereospecific methyl assignments of Val and Leu in OmpX/DHPC. For practical purposes it is of interest that this data could be obtained without use of a deuterated background, and that combinations of NMR experiments have been found for obtaining the desired information either at a 1H frequency of 500 MHz, or with significantly reduced measuring time on a high-frequency instrument.

Bacterial Outer Membrane Proteins↗