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Heinz Rüterjans

Publications and source records attributed to Heinz Rüterjans.

At least 19 recordsLinked to original sources
PubMed2006

Preliminary time-of-flight neutron diffraction study on diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris.

The enzyme diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris is capable of decontaminating a wide variety of toxic organophosphorus nerve agents. DFPase is structurally related to a number of enzymes, such as the medically important paraoxonase (PON). In order to investigate the reaction mechanism of this phosphotriesterase and to elucidate the protonation state of the active-site residues, large-sized crystals of DFPase have been prepared for neutron diffraction studies. Available H atoms have been exchanged through vapour diffusion against D2O-containing mother liquor in the capillary. A neutron data set has been collected to 2.2 A resolution on a relatively small (0.43 mm3) crystal at the spallation source in Los Alamos. The sample size and asymmetric unit requirements for the feasibility of neutron diffraction studies are summarized.

Animals↗
PubMed2006

Binding of a designed substrate analogue to diisopropyl fluorophosphatase: implications for the phosphotriesterase mechanism.

A wide range of organophosphorus nerve agents, including Soman, Sarin, and Tabun is efficiently hydrolyzed by the phosphotriesterase enzyme diisopropyl fluorophosphatase (DFPase) from Loligo vulgaris. To date, the lack of available inhibitors of DFPase has limited studies on its mechanism. The de novo design, synthesis, and characterization of substrate analogues acting as competitive inhibitors of DFPase are reported. The 1.73 A crystal structure of O,O-dicyclopentylphosphoroamidate (DcPPA) bound to DFPase shows a direct coordination of the phosphoryl oxygen by the catalytic calcium ion. The binding mode of this substrate analogue suggests a crucial role for electrostatics in the orientation of the ligand in the active site. This interpretation is further supported by the crystal structures of double mutants D229N/N120D and D229N/N175D, designed to reorient the electrostatic environment around the catalytic calcium. The structures show no differences in their calcium coordinating environment, although they are enzymatically inactive. Additional double mutants E21Q/N120D and E21Q/N175D are also inactive. On the basis of these crystal structures and kinetic and mutagenesis data as well as isotope labeling we propose a new mechanism for DFPase activity. Calcium coordinating residue D229, in concert with direct substrate activation by the metal ion, renders the phosphorus atom of the substrate susceptible for attack of water, through generation of a phosphoenzyme intermediate. Our proposed mechanism may be applicable to the structurally related enzyme paraoxonase (PON), a component of high-density lipoprotein (HDL).

Animals↗
PubMed2006

The electron transfer complex between cytochrome c552 and the CuA domain of the Thermus thermophilus ba3 oxidase. A combined NMR and computational approach.

The structural analysis of the redox complex between the soluble cytochrome c552 and the membrane-integral cytochrome ba3 oxidase of Thermus thermophilus is complicated by the transient nature of this protein-protein interaction. Using NMR-based chemical shift perturbation mapping, however, we identified the contact regions between cytochrome c552 and the CuA domain, the fully functional water-soluble fragment of subunit II of the ba3 oxidase. First we determined the complete backbone resonance assignments of both proteins for each redox state. Subsequently, two-dimensional [15N,1H]TROSY spectra recorded for each redox partner both in free and complexed state indicated those surface residues affected by complex formation between the two proteins. This chemical shift analysis performed for both redox states provided a topological description of the contact surface on each partner molecule. Remarkably, very pronounced indirect effects, which were observed on the back side of the heme cleft only in the reduced state, suggested that alterations of the electron distribution in the porphyrin ring due to formation of the protein-protein complex are apparently sensed even beyond the heme propionate groups. The contact residues of each redox partner, as derived from the chemical shift perturbation mapping, were employed for a protein-protein docking calculation that provided a structure ensemble of 10 closely related conformers representing the complex between cytochrome c552 and the CuA domain. Based on these structures, the electron transfer pathway from the heme of cytochrome c552 to the CuA center of the ba3 oxidase has been predicted.

Cytochrome b Group↗
PubMed2005

Reduced flavin: NMR investigation of N5-H exchange mechanism, estimation of ionisation constants and assessment of properties as biological catalyst.

BACKGROUND: The flavin in its FMN and FAD forms is a versatile cofactor that is involved in catalysis of most disparate types of biological reactions. These include redox reactions such as dehydrogenations, activation of dioxygen, electron transfer, bioluminescence, blue light reception, photobiochemistry (as in photolyases), redox signaling etc. Recently, hitherto unrecognized types of biological reactions have been uncovered that do not involve redox shuffles, and might involve the reduced form of the flavin as a catalyst. The present work addresses properties of reduced flavin relevant in this context. RESULTS: N(5)-H exchange reactions of the flavin reduced form and its pH dependence were studied using the 15N-NMR-signals of 15N-enriched, reduced flavin in the pH range from 5 to 12. The chemical shifts of the N(3) and N(5) resonances are not affected to a relevant extent in this pH range. This contrasts with the multiplicity of the N(5)-resonance, which strongly depends on pH. It is a doublet between pH 8.45 and 10.25 that coalesces into a singlet at lower and higher pH values. From the line width of the 15N(5) signal the pH-dependent rate of hydrogen exchange was deduced. The multiplicity of the 15N(5) signal and the proton exchange rates are little dependent on the buffer system used. CONCLUSION: The exchange rates allow an estimation of the pKa value of N(5)-H deprotonation in reduced flavin to be >or= 20. This value imposes specific constraints for mechanisms of flavoprotein catalysis based on this process. On the other hand the pK asymptotically equal to 4 for N(5)-H protonation (to form N(5)+-H2) would be consistent with a role of N(5)-H as a base.

Air Ionization↗
PubMed2005

Mutational and structural studies of the diisopropylfluorophosphatase from Loligo vulgaris shed new light on the catalytic mechanism of the enzyme.

The active site, the substrate binding site, and the metal binding sites of the diisopropylfluorophosphatase (DFPase) from Loligo vulgaris have been modified by means of site-directed mutagenesis to improve our understanding of the reaction mechanism. Enzymatic characterization of mutants located in the major groove of the substrate binding pocket indicates that large hydrophobic side chains at these positions are favorable for substrate turnover. Moreover, the active site residue His287 proved to be beneficial, but not essential, for DFP hydrolysis. In most cases, hydrophobic side chains at position 287 led to significant catalytic activities although reduced relative to the wild-type enzyme. With respect to the Ca-1 binding site, where catalysis occurs, various mutants indicated that the net charge at this calcium-binding site as well as the relative positions of the charged calcium ligands is crucial for catalytic activity. The importance of the electrostatic potential at the active site was furthermore revealed by various mutations of residues lining the interior of the central water-filled tunnel, which traverses the entire protein structure. In this respect, the structural features of residue His181, which is located at the opposite end of the DFPase tunnel relative to the active site, were characterized extensively. It was concluded that a tunnel-spanning hydrogen bond network, which includes a large number of apparently slow exchanging water molecules, relays any modifications in the electrostatics of the system to the active site, thus affecting the catalytic reactivity of the enzyme.

Animals↗
PubMed2005

Probing the pH-dependent structural features of alpha-KTx12.1, a potassium channel blocker from the scorpion Tityus serrulatus.

Potassium channels are widespread in living cells and are involved in many diseases. The scorpion toxin alpha-KTx(12.1) interacts with various K(+) channels, suggesting its capacity to match diverse channel pores. It is recognized that tissue injuries may affect the pH at toxins site of action, thereby modulating both protein conformation and activity. To better understand its molecular mechanism of action, we studied alpha-KTx(12.1) using pH as a tool to explore its plasticity and NMR in combination with MD calculations to detect it. The toxin solution structure consists of an alpha-helix and a triple-stranded beta-sheet stabilized by four disulfide bridges. The NMR results show, in addition, that His28 possesses an unusually low pK(a) of 5.2. The best set of protein conformers is obtained at pH 4.5, while at pH 7.0, the reduced number of NOEs resulting from a faster hydrogen exchange does not allow to reach a good structural convergence. Nonetheless, MD calculations show that the toxin structure does not vary significantly in that pH range, while conformational changes and modifications of the surface charge distribution occur when His28 is fully protonated. Moreover, essential dynamics analysis reveals variations in the toxin's coherent motions. In conclusion, His28, with its low pK(a) value, provides alpha-KTx(12.1) with the ability to preserve its active conformation over a wide pH interval, thus expanding the range of cellular conditions where the toxin can fully exhibit its activity. Overall, the results further underline the role of histidine as a natural controller of proteins' functionality.

Amino Acid Sequence↗
PubMed2004

Cofactor-apoprotein hydrogen bonding in oxidized and fully reduced flavodoxin monitored by trans-hydrogen-bond scalar couplings.

Hydrogen bonding plays a key role in the tight binding of the FMN cofactor and the regulation of its redox properties in flavodoxins. Hydrogen bonding interactions can be directly observed in solution by multidimensional heteronuclear NMR spectroscopy through the scalar couplings between donor and acceptor nuclei. Here we report on the detection of intermolecular trans-hydrogen-bond couplings ((h)J) between the flavin ring system and the backbone of Desulfovibrio vulgaris flavodoxin in the oxidized and the two-electron reduced states. For this purpose, experiments are adapted from pulse sequences previously applied to determining (h)J coupling constants in nucleic acid-base pairs and proteins. The resulting (h2)J(N,N), (h4)J(N,N), (h3)J(C,N), and (h1)J(H,N) couplings involve the (15)N(1), (13)C(2), and (15)N(3) nuclei of the pyrimidine moiety of FMN, whereas no such interactions are detectable for (13)C(4) and (15)N(5). Several long-range (15)N-(15)N, (13)C-(15)N, and (1)H-(15)N J-coupling constants within the flavin are obtained as "by-products". The magnitudes of both (h)J and regular J couplings are found to be dependent on the redox state. In general, good correlations between (h)J coupling constants and donor-group (1)H chemical shifts and also crystallographic donor-acceptor distances are observed.

Apoproteins↗
PubMed2004

Enhancing bioplastic-substrate interaction via pore induction and directed migration of enzyme location.

We demonstrate two novel approaches to enhance interactions of polymer-immobilized biomolecules with their substrates. In the first approach, diisopropylfluorophosphatase (DFPase) containing poly(urethane) (PU) coatings were made microporous by incorporating, then extracting, poly(ethylene glycol)-based diesters as porogens. Incorporation of 2% w/w porogen increased the effective diffusion coefficient of diisopropylfluorophosphate (DFP) through the coatings by 30% and increased the apparent turnover number of immobilized DFPase 3-fold. In the second approach, prior to immobilization, hydrophobic modification of DFPase was achieved through its conjugation with a dimer/trimer mixture of a uretdione based on 1,6-diisocyanatohexane. When the hydrophobically modified DFPase was immobilized in coatings, catalytic activity was 4-fold higher than that of the equivalent, immobilized, native DFPase. This activity enhancement was independent of the presence or absence of pores. Confocal microscopy images of coatings containing fluorescently labeled lysozyme show that the native enzyme is distributed uniformly over the entire thickness of the coatings. Hydrophobically modified and fluorescently labeled lysozyme is accumulated only in the upper 10 microm cross-sectional layer of a 100 microm-thick coating. Interactions of bioplastics with their substrates are tunable either by pore induction in a polymer or by directed migration of the hydrophobically modified biomolecule to the desired location. The latter approach has broad implications, including overcoming mass transfer limitations experienced by immobilized biocatalysts.

Coated Materials, Biocompatible↗
PubMed2004

Long-range nature of the interactions between titratable groups in Bacillus agaradhaerens family 11 xylanase: pH titration of B. agaradhaerens xylanase.

Xylanase from Bacillus agaradhaerens belongs to a large group of glycosyl hydrolases which catalyze the degradation of xylan. The protonation behavior of titratable groups of the uniformly (15)N- and (13)C-labeled xylanase was investigated by multinuclear NMR spectroscopy. A total of 224 chemical shift titration curves corresponding to (1)H, (13)C, and (15)N resonances revealed pK(a) values for all aspartic and glutamic acid residues, as well as for the C-terminal carboxylate and histidine residues. Most of the titratable groups exhibit a complex titration behavior, which is most likely due to the mutual interactions with other neighboring groups or due to an unusual local microenvironment. Subsite -1 containing the catalytic dyad shows a long-range interaction over 9 A with Asp21 via two hydrogen bonds with Asn45 as the mediator. This result illuminates the pivotal role of the conserved position 45 among family 11 endoxylanases, determining an alkaline pH optimum by asparagine residues or an acidic pH optimum by an aspartate. The asymmetric interactions of neighboring tryptophan side chains with respect to the catalytic dyad can be comprehended as a result of hydrogen bonding and aromatic stacking. Most of the chemical shift-pH profiles of the backbone amides exhibit biphasic behavior with two distinct inflection points, which correspond to the pK(a) values of the nearby acidic side chains. However, the alternation of both positive and negative slopes of individual amide titration curves is interpreted as a consequence of a simultaneous reorganization of side chain conformational space at pH approximately 6 and/or an overall change in the hydrogen network in the substrate binding cleft.

Aspartic Acid↗
PubMed2004

Solution structure of the 30 kDa polysulfide-sulfur transferase homodimer from Wolinella succinogenes.

The periplasmic polysulfide-sulfur transferase (Sud) protein encoded by Wolinella succinogenes is involved in oxidative phosphorylation with polysulfide-sulfur as a terminal electron acceptor. The polysulfide-sulfur is covalently bound to the catalytic Cys residue of the Sud protein and transferred to the active site of the membranous polysulfide reductase. The solution structure of the homodimeric Sud protein has been determined using heteronuclear multidimensional NMR techniques. The structure is based on NOE-derived distance restraints, backbone hydrogen bonds, and torsion angle restraints as well as residual dipolar coupling restraints for a refinement of the relative orientation of the monomer units. The monomer structure consists of a five-stranded parallel beta-sheet enclosing a hydrophobic core, a two-stranded antiparallel beta-sheet, and six alpha-helices. The dimer fold is stabilized by hydrophobic residues and ion pairs found in the contact area between the two monomers. Similar to rhodanese enzymes, Sud catalyzes the transfer of the polysulfide-sulfur to the artificial acceptor cyanide. Despite their similar functions and active sites, the amino acid sequences and structures of these proteins are quite different.

Amino Acid Sequence↗
PubMed2004

Improved method for unambiguous amino acid side-chain 1H and 13C resonance assignment.

Side-chain proton and carbon-13 resonance assignments of [13C;15N]-enriched proteins usually rely on combinations of several multi-dimensional experiments. Here, we describe a four-dimensional pulse sequence, H(C)C-COSY-TOCSY-(CACO)NH, which provides the information required to assign completely aliphatic side-chain resonance frequencies. As in widely used HCC(CO)NH-TOCSY experiments, problems due to spectral crowding are alleviated by exploiting the dispersion of backbone amide 1H and 15N signals. The modification introduced here allows signals from different side-chains to be distinguished even in the case of overlap in the 1H(N)-15N plane of the spectra. For illustration, the new method is applied to two proteins with molecular masses of 11 and 23 kDa.

Algorithms↗
PubMed2004

High level cell-free expression and specific labeling of integral membrane proteins.

We demonstrate the high level expression of integral membrane proteins (IMPs) in a cell-free coupled transcription/translation system using a modified Escherichia coli S30 extract preparation and an optimized protocol. The expression of the E. coli small multidrug transporters EmrE and SugE containing four transmembrane segments (TMS), the multidrug transporter TehA with 10 putative TMS, and the cysteine transporter YfiK with six putative TMS, were analysed. All IMPs were produced at high levels yielding up to 2.7 mg of protein per mL of reaction volume. Whilst the vast majority of the synthesized IMPs were precipitated in the reaction mixture, the expression of a fluorescent EmrE-sgGFP fusion construct showed evidence that a small part of the synthesized protein 'remained soluble and this amount could be significantly increased by the addition of E. coli lipids into the cell-free reaction. Alternatively, the majority of the precipitated IMPs could be solubilized in detergent micelles, and modifications to the solubilization procedures yielded proteins that were almost pure. The folding induced by formation of the proposed alpha-helical secondary structures of the IMPs after solubilization in various micelles was monitored by CD spectroscopy. Furthermore, the reconstitution of EmrE, SugE and TehA into proteoliposomes was demonstrated by freeze-fracture electron microscopy, and the function of EmrE was additionally analysed by the specific transport of ethidium. The cell-free expression technique allowed efficient amino acid specific labeling of the IMPs with 15N isotopes, and the recording of solution NMR spectra of the solubilized EmrE, SugE and YfiK proteins further indicated a correctly folded conformation of the proteins.

Base Sequence↗
PubMed2003

Statistical analysis of crystallographic data obtained from squid ganglion DFPase at 0.85 A resolution.

The X-ray crystal structure of squid-type diisopropylfluorophosphatase (DFPase) has been refined to a resolution of 0.85 A and a crystallographic R value of 9.4%. Crystal annealing improved both the mosaicity and resolution of the crystals considerably. The overall structure of this protein represents a six-bladed beta-propeller with two calcium ions bound in a central water-filled tunnel. 496 water, two glycerol and two MES buffer molecules and 18 PEG fragments of different lengths could be refined in the solvent region. 45 of the 314 residues have been refined with alternative orientations. H atoms have been omitted from disordered residues. For the residues of the inner beta-strands, H atoms are visible in a normal F(o) - F(c) difference map of a hydrogen-deficient structure model. The 208 most reliable residues, without disorder or reduced occupancy in their side chains, were finally refined without restraints. A subsequent full-matrix refinement cycle for the positional parameters yielded estimated standard deviations (e.s.d.s) by matrix inversion. The thus calculated bond lengths and bond angles and their e.s.d.s were used to obtain averaged bond lengths and bond angles, which were compared with the restraints applied in the preceding refinement cycles. The lengths and angles of the hydrogen bonds inside the antiparallel beta-sheets of the DFPase structure were compared with data averaged over 11 high-resolution protein structures. Torsion angles were averaged according to angle types used as restraints in X-PLOR and CNS and subsequently compared with values obtained from 46 high-resolution structures. Side-chain torsion angles were also classified into rotamer types according to the Penultimate Rotamer Library. Moreover, precise dimensions for both Ca(2+)-coordination polyhedra could be obtained and the coordination of one Ca(2+) ion by an imidazole N atom was confirmed. This statistical analysis thus provides a first step towards a set of restraints that are founded completely on macromolecular data; however, 10-20 additional protein data sets of comparable accuracy and size will be required to obtain a larger statistical base, especially for side-chain analysis.

Amino Acids↗
PubMed2003

Interaction of cytochrome c with cytochrome c oxidase: an NMR study on two soluble fragments derived from Paracoccus denitrificans.

The functional interactions between the various components of the respiratory chain are relatively short-lived, thus allowing high turnover numbers but at the same time complicating the structural analysis of the complexes. Chemical shift mapping by NMR spectroscopy is a useful tool to investigate such transient contacts, since it can monitor changes in the electron-shielding properties of a protein as the result of temporary contacts with a reaction partner. In this study, we investigated the molecular interaction between two components of the electron-transfer chain from Paracoccus denitrificans: the engineered, water-soluble fragment of cytochrome c(552) and the Cu(A) domain from the cytochrome c oxidase. Comparison of [(15)N,(1)H]-TROSY spectra of the [(15)N]-labeled cytochrome c(552) fragment in the absence and in the presence of the Cu(A) fragment showed chemical shift changes for the backbone amide groups of several, mostly uncharged residues located around the exposed heme edge in cytochrome c(552). The detected contact areas on the cytochrome c(552) surface were comparable under both fully reduced and fully oxidized conditions, suggesting that the respective chemical shift changes represent biologically relevant protein-protein interactions.

Bacterial Proteins↗
PubMed2003

Structural analysis of the DNA-binding domain of the Erwinia amylovora RcsB protein and its interaction with the RcsAB box.

The transcriptional regulator RcsB interacts with other coactivators to control the expression of biosynthetic operons in enterobacteria. While in a heterodimer complex with the regulator RcsA the RcsAB box consensus is recognized, DNA binding sites for RcsB without RcsA have also been identified. The conformation of RcsB might therefore be modulated upon interaction with various coactivators, resulting in the recognition of different DNA targets. We report the solution structure of the C-terminal DNA-binding domain of the RcsB protein from Erwinia amylovora spanning amino acid residues 129-215 solved by heteronuclear magnetic resonance (NMR) spectroscopy. The C-terminal domain is composed of four alpha-helices where two central helices form a helix-turn-helix motif similar to the structures of the regulatory proteins GerE, NarL, and TraR. Amino acid residues involved in the RcsA independent DNA binding of RcsB were identified by titration studies with a RcsAB box consensus fragment. Data obtained from NMR spectroscopy together with surface plasmon resonance measurements demonstrate that the RcsAB box is specifically recognized by the RcsAB heterodimer as well as by RcsB alone. However, the binding constant of RcsB alone at target promoters from Escherichia coli, E. amylovora, and Pantoea stewartii was approximately 1 order of magnitude higher compared with that of the RcsAB heterodimer. We present evidence that the obvious role of RcsA is not to alter the DNA binding specificity of RcsB but to stabilize RcsB-DNA complexes.

Amino Acid Motifs↗
PubMed2003

Simultaneous measurement of protein one-bond and two-bond nitrogen-carbon coupling constants using an internally referenced quantitative J-correlated [(15)N,(1)H]-TROSY-HNC experiment.

A quantitative J-correlation pulse sequence is described that allows simultaneous determination of one-bond and two-bond nitrogen-carbon coupling constants for protonated or deuterated proteins. Coupling constants are calculated from volume ratios between cross peaks and reference axial peaks observed in a single 3D spectrum. Accurate backbone (1)J(NC'), (1)J(NCalpha), and (2)J(NCalpha) coupling constants are obtained for the two [(15)N;(13)C]-labeled, medium-sized proteins flavodoxin and xylanase and for the [(2)H;(15)N;(13)C]-labeled, large protein DFPase. A dependence of one-bond and two-bond J(NCalpha) values on protein backbone psi torsion angles is readily apparent, in agreement with previously found correlations. In addition, the experiment is performed on isotropic as well as aligned protein to measure associated (15)N-(13)C residual dipolar couplings.

Bacillus↗