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Eike Brunner

Publications and source records attributed to Eike Brunner.

17 recordsLinked to original sources
PubMed2006

Rapid assignment of solution 31P NMR spectra of large proteins by solid-state spectroscopy.

The application of the (31)P NMR spectroscopy to large proteins or protein complexes in solution is hampered by a relatively low intrinsic sensitivity coupled with large line widths. Therefore, the assignment of the phosphorus signals by two-dimensional NMR methods in solution is often extremely time consuming. In contrast, the quality of solid-state NMR spectra is not dependent on the molecular mass and the solubility of the protein. For the complex of Ras with the GTP-analogue GppCH(2)p we show solid-state (31)P NMR methods to be more sensitive by almost one order of magnitude than liquid-state NMR. Thus, solid-state NMR seems to be the method of choice for obtaining the resonance assignment of the phosphorus signals of protein complexes in solution. Experiments on Ras.GDP complexes show that the microcrystalline sample can be substituted by a precipitate of the sample and that unexpectedly the two structural states observed earlier in solution are present in crystals as well.

Crystallization↗
PubMed2005

NMR spectroscopic study of noble gas binding into the engineered cavity of HPr(I14A) from Staphylococcus carnosus.

Xenon binding into preexisting cavities in proteins is a well-known phenomenon. Here we investigate the interaction of helium, neon, and argon with hydrophobic cavities in proteins by NMR spectroscopy. 1H and 15N chemical shifts of the I14A mutant of the histidine-containing phosphocarrier protein (HPr(I14A)) from Staphylococcus carnosus are analyzed by chemical shift mapping. Total noble gas induced chemical shifts, Delta, are calculated and compared with the corresponding values obtained using xenon as a probe atom. This comparison reveals that the same cavity is detected with both argon and xenon. Measurements using the smaller noble gases helium and neon as probe atoms do not result in comparable effects. The dependence of amide proton and nitrogen chemical shifts on the argon concentration is investigated in the range from 10 mM up to 158 mM. The average dissociation constant for argon binding into the engineered cavity is determined to be about 90 mM.

Amino Acids↗
PubMed2005

Biomineralization in diatoms: characterization of novel polyamines associated with silica.

Pattern formation during silica biomineralization in diatoms appears to depend on long-chain polyamines as well as proteins covalently modified with polyamines (silaffins). Recently, the complete genome of the diatom Thalassiosira pseudonana has been sequenced making this species an attractive model organism for future studies on biomineralization. Mass- and NMR-spectroscopic analysis of the long-chain polyamines from this diatom species reveals the existence of a complex population with as yet unknown structural features. These include complex methylation patterns, different attachment moieties as well as the existence of quaternary ammonium functionalities.

Diatoms↗
PubMed2005

Biomimetic silica formation: analysis of the phosphate-induced self-assembly of polyamines.

The highly siliceous cell walls of diatoms are probably the most outstanding examples of nanostructured materials in nature. Previous in vitro experiments have shown that the biomolecules found in the cell walls of diatoms, namely polyamines and silaffins, are capable of catalysing the formation of silica nanospheres from silicic/oligosilicic acid solutions. In a previous publication, silica precipitation was found to be strictly correlated with a phosphate-induced microscopic phase separation of the polyamines. The present contribution further characterises the phase separation behaviour of polyamines in aqueous solutions. In particular, a pronounced pH-dependence of the average particle diameter is found. It is, furthermore, shown that the ability of phosphate ions to form polyamine aggregates in aqueous solutions cannot be a purely electrostatic effect. Instead, a defined hydrogen-bonded network stabilised by properly balanced electrostatic interactions should be considered. Finally, solid-state 31P NMR studies on phase-separated polyamines, synthetic silica precipitates, and diatom cell walls from the species Coscinodicus granii support the assumption of a phosphate-induced phase separation process taking place during cell wall formation.

Biomimetic Materials↗
PubMed2005

Reverse micelles dissolved in supercritical xenon: an NMR spectroscopic study.

Reverse micelles currently gain increasing interest in chemical technology. They also become important in biomolecular NMR due to their ability to host biomolecules such as proteins. In the present paper, a procedure for the preparation of high-pressure NMR samples containing reverse micelles dissolved in supercritical xenon is presented. These reverse micelles are formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT). For the first time, NMR spectroscopy could be applied to reverse micelles in supercritical xenon. The AOT/H(2)O/Xe system was studied as a function of experimental parameters such as xenon pressure, water content, and salt concentration. Optimum conditions for reverse micelle formation in supercritical xenon could be determined. It is, furthermore, demonstrated that biomolecules such as amino acids and proteins can be incorporated into the reverse micelles dissolved in supercritical xenon.

Chemistry, Physical↗
PubMed2004

Solid-state 31P NMR spectroscopy of microcrystals of the Ras protein and its effector loop mutants: comparison between crystalline and solution state.

Cycling between a GTP bound "on" state and a GDP bound "off" state, guanine nucleotide-binding (GNB) proteins act as molecular switches. The switching process and the interaction with effectors, GTPase-activating proteins, and guanosine nucleotide-exchange factors is accompanied by pronounced conformational changes of the switch regions of the GNB proteins. The aim of the present contribution is to correlate conformational changes observed by liquid-state NMR with solid-state (31)P NMR data and with the results of X-ray crystallography. Crystalline wild-type Ras complexed with GTP analogs such as GppCH(2)p and GppNHp could be prepared. At low temperatures, two different signals were found for the gamma-phosphate group of GppNHp bound to wild-type Ras. This behavior indicates the existence of two different conformations of the molecule in the crystalline state as it is found in solution but not by X-ray crystallography. In contrast to the GppNHp complex, the two separate gamma-phosphate signals could not be observed for GppCH(2)p bound to wild-type Ras. However, an increasing linewidth at low temperature indicates the presence of an exchange process. The results obtained for the wild-type protein are compared with the behavior of GppNHp complexes of the effector loop mutants Ras(T35S) and Ras(T35A). These mutants prefer a conformation similar to the GDP bound "off" state.

Binding Sites↗
PubMed2004

Phosphorylated amino acids: model compounds for solid-state 31P NMR spectroscopic studies of proteins.

Solid-state 31P NMR spectroscopy was applied to measure the isotropic chemical shifts, chemical shift anisotropies and asymmetry parameters of three phosphorylated amino acids, O-phospho-L-serine, O-phospho-L-threonine and O-phospho-L-tyrosine. The cross-polarization buildup rates and longitudinal relaxation times of 31P and 1H were-determined and compared with the values measured for a triphosphate (GppCH2p) bound to a crystalline protein (Ras). It is shown that the phosphorylated amino acids are well-suited model compounds, e.g. for the optimization of experiments on crystalline proteins. Two-dimensional exchange experiments on O-phospho-L-tyrosine indicate the existence of an exchange between the two different conformations of the molecule.

Amino Acids↗
PubMed2004

High-temperature solution NMR structure of TmCsp.

Cold shock proteins (Csps) are assumed to play a central role in the regulation of gene expression under cold shock conditions. Acting as single-stranded nucleic acid-binding proteins, they trigger the translation process and are therefore involved in the compensation of the influence of low temperatures (cold shock) upon the cell metabolism. However, it is unknown so far how Csps are switched on and off as a function of temperature. The aim of the present study is the study of possible structural changes responsible for this switching process. (1)H-(15)N HSQC spectra recorded at different temperatures and chemical-shift analysis have indicated subtle conformational changes for the cold-shock protein from the hyperthermophilic bacterium Thermotoga maritima (TmCsp) when the temperature is elevated from 303 K to its physiological temperature (343 K). The three-dimensional structure of TmCsp was determined by nuclear magnetic resonance (NMR) spectroscopy at 343 K to obtain quantitative information concerning these structural changes. By use of residual dipolar couplings, the loss of NOE information at high temperature could be compensated successfully. Most pronounced conformational changes compared with room-temperature conditions are observed for amino acid residues closely neighbored to two characteristic beta-bulges and a well-defined loop region of the protein. Because the residues shown to be responsible for the interaction of TmCsp with single-stranded nucleic acids can almost exclusively be found within these regions, nucleic acid-binding activity might be down-regulated with increasing temperature by the described conformational changes.

Bacterial Proteins↗
PubMed2004

Solution structure of the active-centre mutant I14A of the histidine-containing phosphocarrier protein from Staphylococcus carnosus.

High-pressure NMR experiments performed on the histidine-containing phosphocarrier protein (HPr) from Staphylococcus carnosus have shown that residue Ile14, which is located in the active-centre loop, exhibits a peculiarly small pressure response. In contrast, the rest of the loop shows strong pressure effects as is expected for typical protein interaction sites. To elucidate the structural role of this residue, the mutant protein HPr(I14A), in which Ile14 is replaced by Ala, was produced and studied by solution NMR spectroscopy. On the basis of 1406 structural restraints including 20 directly detected hydrogen bonds, 49 1H(N)-15N, and 25 1H(N)-1Halpha residual dipolar couplings, a well resolved three-dimensional structure could be determined. The overall fold of the protein is not influenced by the mutation but characteristic conformational changes are introduced into the active-centre loop. They lead to a displacement of the ring system of His15 and a distortion of the N-terminus of the first helix, which supports the histidine ring. In addition, the C-terminal helix is bent because the side chain of Leu86 located at the end of this helix partly fills the hydrophobic cavity created by the mutation. Xenon, which is known to occupy hydrophobic cavities, causes a partial reversal of the mutation-induced structural effects. The observed structural changes explain the reduced phosphocarrier activity of the mutant and agree well with the earlier suggestion that Ile14 represents an anchoring point stabilizing the active-centre loop in its correct conformation.

Bacterial Proteins↗
PubMed2003

NMR-spectroscopic mapping of an engineered cavity in the I14A mutant of HPr from Staphylococcus carnosus using xenon.

The interaction between the histidine-containing phosphocarrier protein HPr and xenon atoms in solution is studied in the present paper. Wild-type HPr as well as the exchange mutant I14A have been studied. Specific binding of xenon into an engineered cavity created via the exchange of amino acid residue I14 by alanine could be shown using 1H-15N heteronuclear single-quantum coherence (HSQC) spectroscopy. Xenon binding results in pronounced changes of the 1H and 15N chemical shifts of amide groups close to the cavity. In addition to this observation which allows the NMR-spectroscopic mapping of such cavities, we have shown that the entire molecule is slightly rearranged as a result of xenon binding. In contrast, wild-type HPr only exhibits minor chemical shift changes due to the nonspecific interactions with the xenon atoms in solution.

Alanine↗
PubMed2003

Solution structure of the matrix attachment region-binding domain of chicken MeCP2.

Methyl-CpG-binding protein 2 (MeCP2) is a multifunctional protein involved in chromatin organization and silencing of methylated DNA. MAR-BD, a 125-amino-acid residue domain of chicken MeCP2 (cMeCP2, originally named ARBP), is the minimal protein fragment required to recognize MAR elements and mouse satellite DNA. Here we report the solution structure of MAR-BD as determined by multidimensional heteronuclear NMR spectroscopy. The global fold of this domain is very similar to that of rat MeCP2 MBD and MBD1 MBD (the methyl-CpG-binding domains of rat MeCP2 and methyl-CpG-binding domain protein 1, respectively), exhibiting a three-stranded antiparallel beta-sheet and an alpha-helix alpha1. We show that the C-terminal portion of MAR-BD also contains an amphipathic helical coil, alpha2/alpha3. The hydrophilic residues of this coil form a surface opposite the DNA interface, available for interactions with other domains of MeCP2 or other proteins. Spectroscopic studies of the complex formed by MAR-BD and a 15-bp fragment of a high-affinity binding site from mouse satellite DNA indicates that the coil is also involved in protein.DNA interactions. These studies provide a basis for discussion of the consequences of six missense mutations within the helical coil found in Rett syndrome cases.

Animals↗
PubMed2002

Self-assembly of highly phosphorylated silaffins and their function in biosilica morphogenesis.

Silaffins are uniquely modified peptides that have been implicated in the biogenesis of diatom biosilica. A method that avoids the harsh anhydrous hydrogen fluoride treatment commonly used to dissolve biosilica allows the extraction of silaffins in their native state. The native silaffins carry further posttranslational modifications in addition to their polyamine moieties. Each serine residue was phosphorylated, and this high level of phosphorylation is essential for biological activity. The zwitterionic structure of native silaffins enables the formation of supramolecular assemblies. Time-resolved analysis of silica morphogenesis in vitro detected a plastic silaffin-silica phase, which may represent a building material for diatom biosilica.

Cell Wall↗
PubMed2002

Determination of residual dipolar couplings in homonuclear MOCCA-SIAM experiments.

In solutions with partial molecular alignment, anisotropic magnetic interactions such as the chemical shift anisotropy, the electric quadrupole interaction, and the magnetic dipole-dipole interaction are no longer averaged out to zero in contrast to isotropic solutions. The resulting residual anisotropic magnetic interactions are increasingly used in biological NMR studies for the determination of 3D structures of proteins and other biomolecules. In the present paper we propose a new approach allowing the measurement of residual HN-H(alpha) dipolar couplings of non-isotope enriched proteins based on the application of the MOCCA-SIAM experiment. This experiment allows the measurement of homonuclear coupling constants with an accuracy of ca. +/- 0.2 Hz and is therefore particularly well suited to determine residual dipolar couplings at relatively low degrees of molecular orientation. The agreement between experimentally determined residual HN-H(alpha) couplings and calculated values is demonstrated for BPTI.

Animals↗