Jürgen Schleucher

Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients

Heteronuclear multidimensional NMR experiments for the structure determination of proteins in solution employing pulsed field gradients

A general enhancement scheme in heteronuclear multidimensional NMR employing pulsed field gradients.

SummaryGeneral pulse sequence elements that achieve sensitivity-enhanced coherence transfer from a heteronucleus to protons of arbitrary multiplicity are introduced. The building blocks are derived from the sensitivity-enhancement scheme introduced by Cavanagh et al. ((1991) J. Magn. Reson., 91, 429–436), which was used in conjunction with gradient coherence selection by Kay et al. ((1992) J. Am. Chem. Soc., 114, 10663–10665), as well as from a multiple-pulse sequence effecting a heteronuclear planar coupling Hamiltonian. The building blocks are incorporated into heteronuclear correlation experiments, in conjunction with coherence selection by the formation of a heteronuclear gradient echo. This allows for efficient water suppression without the need for water presaturation. The methods are demonstrated in HSQC-type experiments on a sample of a decapeptide in H2O. The novel pulse sequence elements can be incorporated into multidimensional experiments.

Biochemical and functional characterization of Helicobacter pylori vesicles.

Helicobacter pylori can cause peptic ulcer disease and/or gastric cancer. Adhesion of bacteria to the stomach mucosa is an important contributor to the vigour of infection and resulting virulence. H. pylori adheres primarily via binding of BabA adhesins to ABO/Lewis b (Leb) blood group antigens and the binding of SabA adhesins to sialyl‐Lewis x/a (sLex/a) antigens. Similar to most Gram‐negative bacteria, H. pylori continuously buds off vesicles and vesicles derived from pathogenic bacteria often include virulence‐associated factors. Here we biochemically characterized highly purified H. pylori vesicles. Major protein and phospholipid components associated with the vesicles were identified with mass spectroscopy and nuclear magnetic resonance. A subset of virulence factors present was confirmed by immunoblots. Additional functional and biochemical analysis focused on the vesicle BabA and SabA adhesins and their respective interactions to human gastric epithelium. Vesicles exhibit heterogeneity in their protein composition, which were specifically studied in respect to the BabA adhesin. We also demonstrate that the oncoprotein, CagA, is associated with the surface of H. pylori vesicles. Thus, we have explored mechanisms for intimate H. pylori vesicle–host interactions and found that the vesicles carry effector‐promoting properties that are important to disease development.

Probing solvent accessibility of amyloid fibrils by solution NMR spectroscopy

Amyloid is the result of an anomalous protein and peptide aggregation, leading to the formation of insoluble fibril deposits. At present, 18 human diseases have been associated with amyloid deposits—e.g., Alzheimers disease and Prion-transmissible Spongiform Encephalopathies. The molecular structure of amyloid is to a large extent unknown, because of lack of high-resolution structural information within the amyloid state. However, from other experimental data it has been established that amyloid fibrils predominantly consist of β-strands arranged perpendicular to the fibril axis. Identification of residues involved in these secondary structural elements is therefore of vital importance to rationally designing appropriate inhibitors. We have designed a hydrogen/deuterium exchange NMR experiment that can be applied on mature amyloid to enable identification of the residues located inside the fibril core. Using a highly amyloidogenic peptide, corresponding to residues 25–35 within the Alzheimer Aβ(1–43) peptide, we could establish that residues 28–35 constitute the amyloid core, with residues 31 and 32 being the most protected. In addition, quantitative values for the solvent accessibility for each involved residue could be obtained. Based on our data, two models of peptide assembly are proposed. The method provides a general way to identify the core of amyloid structures and thereby pinpoint areas suitable for design of inhibitors.

A simultaneous 15N,1H- and 13C,1H-HSQC with sensitivity enhancement and a heteronuclear gradient echo

SummaryNew pulse sequences are introduced and discussed that allow for simultaneous acquisition of 15N,1H-and 13C,1H-HSQC correlations for fully 13C,15N-labeled biomacromolecules in combination with hetero-nuclear gradient echoes and sensitivity enhancement. The pulse sequence experimentally found to be optimal can be used as a building block, especially in time-consuming multidimensional NMR experiments. Due to the excellent solvent suppression obtained by employing heteronuclear gradient echoes, which allows detection of resonances under the water resonance, it would be possible to record two sensitivity-enhanced 4D experiments simultaneously on one sample dissolved in H2O, e.g. a 4D 13C,1H-HSQC-NOESY-15N, 1H/13C,1H-HSQC.

Mutational and Structural Analyses of the Ribonucleotide Reductase Inhibitor Sml1 Define Its Rnr1 Interaction Domain Whose Inactivation Allows Suppression of mec1 and rad53 Lethality

ABSTRACT In budding yeast, MEC1 and RAD53 are essential for cell growth. Previously we reported that mec1or rad53 lethality is suppressed by removal of Sml1, a protein that binds to the large subunit of ribonucleotide reductase (Rnr1) and inhibits RNR activity. To understand further the relationship between this suppression and the Sml1-Rnr1 interaction, we randomly mutagenized the SML1 open reading frame. Seven mutations were identified that did not affect protein expression levels but relieved mec1 and rad53inviability. Interestingly, all seven mutations abolish the Sml1 interaction with Rnr1, suggesting that this interaction causes the lethality observed in mec1 and rad53strains. The mutant residues all cluster within the 33 C-terminal amino acids of the 104-amino-acid-long Sml1 protein. Four of these residues reside within an alpha-helical structure that was revealed by nuclear magnetic resonance studies. Moreover, deletions encompassing the N-terminal half of Sml1 do not interfere with its RNR inhibitory activity. Finally, the seven sml1 mutations also disrupt the interaction with yeast Rnr3 and human R1, suggesting a conserved binding mechanism between Sml1 and the large subunit of RNR from different species.

Both catabolic and anabolic heterotrophic microbial activity proceed in frozen soils

A large proportion of the global soil carbon pool is stored in soils of high-latitude ecosystems in which microbial processes and production of greenhouse gases proceed during the winter months. It has been suggested that microorganisms have limited ability to sequester substrates at temperatures around and below 0 °C and that a metabolic shift to dominance of catabolic processes occurs around these temperatures. However, there are contrary indications that anabolic processes can proceed, because microbial growth has been observed at far lower temperatures. Therefore, we investigated the utilization of the microbial substrate under unfrozen and frozen conditions in a boreal forest soil across a temperature range from −9 °C to +9 °C, by using gas chromatography-isotopic ratio mass spectrometry and 13C magic-angle spinning NMR spectroscopy to determine microbial turnover and incorporation of 13C-labeled glucose. Our results conclusively demonstrate that the soil microorganisms maintain both catabolic (CO2 production) and anabolic (biomass synthesis) processes under frozen conditions and that no significant differences in carbon allocation from [13C]glucose into [13C]CO2 and cell organic 13C-compounds occurred between +9 °C and −4 °C. The only significant metabolic changes detected were increased fluidity of the cell membranes synthesized at frozen conditions and increased production of glycerol in the frozen samples. The finding that the processes in frozen soil are similar to those in unfrozen soil has important implications for our general understanding and conceptualization of soil carbon dynamics in high-latitude ecosystems.

Protein oligomerization induced by oleic acid at the solid–liquid interface – equine lysozyme cytotoxic complexes

Protein oligomeric complexes have emerged as a major target of current research because of their key role in aggregation processes in living systems and in vitro. Hydrophobic and charged surfaces may favour the self‐assembly process by recruiting proteins and modifying their interactions. We found that equine lysozyme assembles into multimeric complexes with oleic acid (ELOA) at the solid–liquid interface within an ion‐exchange chromatography column preconditioned with oleic acid. The properties of ELOA were characterized using NMR, spectroscopic methods and atomic force microscopy, and showed similarity with both amyloid oligomers and the complexes with oleic acid and its structural homologous protein α‐lactalbumin, known as humanα‐lactalbumin made lethal for tumour cells (HAMLET). As determined by NMR diffusion measurements, ELOA may consist of 4–30 lysozyme molecules. Each lysozyme molecule is able to bind 11–48 oleic acids in various preparations. Equine lysozyme acquired a partially unfolded conformation in ELOA, as evident from its ability to bind hydrophobic dye 8‐anilinonaphthalene‐1‐sulfonate. CD and NMR spectra. Similar to amyloid oligomers, ELOA also interacts with thioflavin‐T dye, shows a spherical morphology, assembles into ring‐shaped structures, as monitored by atomic force microscopy, and exerts a toxic effect in cells. Studies of well‐populated ELOA shed light on the nature of the amyloid oligomers and HAMLET complexes, suggesting that they constitute one large family of cytotoxic proteinaceous species. The hydrophobic surfaces can be used profitably to produce complexes with very distinct properties compared to their precursor proteins.

Novel strategies for sensitivity enhancement in heteronuclear multi—dimensional NMR experiments employing pulsed field gradients

SummaryNovel strategies for sensitivity enhancement in heteronuclear multidimensional spectra are introduced and evaluated theoretically and experimentally. It is shown that in 3D sequences employing several Coherence Order Selective Coherence Transfer (COS-CT) steps, enhancement factors of up to 2 can be achieved. This sensitivity enhancement is compatible with the use of heteronuclear gradient echoes, yielding spectra with excellent water suppression. HNCO and HCCH-TOCSY pulse sequences are proposed and experimentally tested. These experiments employ recently developed coherence order selective pulse sequence elements, e.g., COS-INEPT and planar TOCSY for antiphase to in-phase transfers 2F-S2↔S- or in-phaseaCOS-CT for in-phase transfer F-↔S-, and the well-known isotropic TOCSY mixing sequences for homo- and heteronuclear in-phase transfer.

High-resolution characterization of organic phosphorus in soil extracts using 2D 1H-31P NMR correlation spectroscopy.

Organic phosphorus (P) compounds represent a major component of soil P in many soils and are key sources of P for microbes and plants. Solution NMR (nuclear magnetic resonance spectroscopy) is a powerful technique for characterizing organic P species. However, (31)P NMR spectra are often complicated by overlapping peaks, which hampers identification and quantification of the numerous P species present in soils. Overlap is often exacerbated by the presence of paramagnetic metal ions, even if they are in complexes with EDTA following NaOH/EDTA extraction. By removing paramagnetic impurities using a new precipitation protocol, we achieved a dramatic improvement in spectral resolution. Furthermore, the obtained reduction in line widths enabled the use of multidimensional NMR methods to resolve overlapping (31)P signals. Using the new protocol on samples from two boreal humus soils with different Fe contents, 2D (1)H-(31)P correlation spectra allowed unambiguous identification of a large number of P species based on their (31)P and (1)H chemical shifts and their characteristic coupling patterns, which would not have been possible using previous protocols. This approach can be used to identify organic P species in samples from both terrestrial and aquatic environments increasing our understanding of organic P biogeochemistry.