Ute C. Marx

The Metabolome of Chlamydomonas reinhardtii following Induction of Anaerobic H2 Production by Sulfur Depletion

The metabolome of the model species Chlamydomonas reinhardtii has been analyzed during 120 h of sulfur depletion to induce anaerobic hydrogen (H2) production, using NMR spectroscopy, gas chromatography coupled to mass spectrometry, and TLC. The results indicate that these unicellular green algae consume freshly supplied acetate in the medium to accumulate energy reserves during the first 24 h of sulfur depletion. In addition to the previously reported accumulation of starch, large amounts of triacylglycerides were deposited in the cells. During the early 24- to 72-h time period fermentative energy metabolism lowered the pH, H2 was produced, and amino acid levels generally increased. In the final phase from 72 to 120 h, metabolism slowed down leading to a stabilization of pH, even though some starch and most triacylglycerides remained. We conclude that H2 production does not slow down due to depletion of energy reserves but rather due to loss of essential functions resulting from sulfur depletion or due to a build-up of the toxic fermentative products formate and ethanol.

Receptor recognition by a hepatitis B virus reveals a novel mode of high affinity virus–receptor interaction

The duck hepatitis B virus model system was used to elucidate the characteristics of receptor (carboxypeptidase D, gp180) interaction with polypeptides representing the receptor binding site in the preS part of the large viral surface protein. We demonstrate the pivotal role of carboxypeptidase D for virus entry and show its C‐domain represents the virus attachment site, which binds preS with extraordinary affinity. Combining results from surface plasmon resonance spectroscopy and two‐dimensional NMR analysis we resolved the contribution of preS sequence elements to complex stability and show that receptor binding potentially occurs in two steps. Initially, a short α‐helix in the C‐terminus of the receptor binding domain facilitates formation of a primary complex. This complex is stabilized sequentially, involving ∼60 most randomly structured amino acids preceding the helix. Thus, hepadnaviruses exhibit a novel mechanism of high affinity receptor interaction by conserving the potential to adapt structure during binding rather than to preserve it per se. We propose that this process represents an alternative strategy to escape immune surveillance and the evolutionary pressure inherent in the compact hepadnaviral genome organization.

CD and NMR Studies of Prion Protein (PrP) Helix 1 NOVEL IMPLICATIONS FOR ITS ROLE IN THE PrPC→ PrPSc CONVERSION PROCESS

The conversion of prion helix 1 from an α-helical into an extended conformation is generally assumed to be an essential step in the conversion of the cellular isoform PrPC of the prion protein to the pathogenic isoform PrPSc. Peptides encompassing helix 1 and flanking sequences were analyzed by nuclear magnetic resonance and circular dichroism. Our results indicate a remarkably high instrinsic helix propensity of the helix 1 region. In particular, these peptides retain significant helicity under a wide range of conditions, such as high salt, pH variation, and presence of organic co-solvents. As evidenced by a data base search, the pattern of charged residues present in helix 1 generally favors helical structures over alternative conformations. Because of its high stability against environmental changes, helix 1 is unlikely to be involved in the initial steps of the pathogenic conformational change. Our results implicate that interconversion of helix 1 is rather representing a barrier than a nucleus for the PrPC→ PrPSc conversion.

Phylogenetic and molecular analysis of hydrogen-producing green algae

A select set of microalgae are reported to be able to catalyse photobiological H2 production from water. Based on the model organism Chlamydomonas reinhardtii, a method was developed for the screening of naturally occurring H2-producing microalgae. By purging algal cultures with N2 in the dark and subsequent illumination, it is possible to rapidly induce photobiological H2 evolution. Using NMR spectroscopy for metabolic profiling in C. reinhardtii, acetate, formate, and ethanol were found to be key compounds contributing to metabolic variance during the assay. This procedure can be used to test algal species existing as axenic or mixed cultures for their ability to produce H2. Using this system, five algal isolates capable of H2 production were identified in various aquatic systems. A phylogenetic tree was constructed using ribosomal sequence data of green unicellular algae to determine if there were taxonomic patterns of H2 production. H2-producing algal species were seen to be dispersed amongst most clades, indicating an H2-producing capacity preceded evolution of the phylum Chlorophyta.

The structure of human parathyroid hormone-related protein(1-34) in near-physiological solution.

Parathyroid hormone‐related protein plays a major role in the pathogenesis of humoral hypercalcemia of malignancy. Under normal physiological conditions, parathyroid hormone‐related protein is produced in a wide variety of tissues and acts in an autocrine or paracrine fashion. Parathyroid hormone‐related protein and parathyroid hormone bind to and activate the same G‐protein‐coupled receptor. Here we present the structure of the biologically active NH2‐terminal domain of human parathyroid hormone‐related protein(1–34) in near‐physiological solution in the absence of crowding reagents as determined by two‐dimensional proton magnetic resonance spectroscopy. An improved strategy for structure calculation revealed the presence of two helices, His‐5–Leu‐8 and Gln‐16–Leu‐27, connected by a flexible linker. The parathyroid hormone‐related protein(1–34) structure and the structure of human parathyroid hormone(1–37) as well as human parathyroid hormone(1–34) are highly similar, except for the well defined turn, His‐14–Ser‐17, present in parathyroid hormone. Thus, the similarity of the binding affinities of parathyroid hormone and parathyroid hormone‐related protein to their common receptor may be based on their structural similarity.

Solution Structure of Human Proguanylin THE ROLE OF A HORMONE PROSEQUENCE

The endogenous ligand of guanylyl cyclase C, guanylin, is produced as the 94-amino-acid prohormone proguanylin, with the hormone guanylin located at the COOH terminus of the prohormone. The solution structure of proguanylin adopts a new protein fold and consists of a three-helix bundle, a small three-stranded β-sheet of two NH2-terminal strands and one COOH-terminal strand, and an unstructured linker region. The sequence corresponding to guanylin is fixed in its bioactive topology and is involved in interactions with the NH2-terminal β-hairpin: the hormone region (residues 80–94) partly wraps around the first 4 NH2-terminal residues that thereby shield parts of the hormone surface. These interactions provide an explanation for the negligible bioactivity of the prohormone as well as the important role of the NH2-terminal residues in the disulfide-coupled folding of proguanylin. Since the ligand binding region of guanylyl cyclase C is predicted to be located around an exposed β-strand, the intramolecular interactions observed between guanylin and its prosequence may be comparable with the guanylin/receptor interaction.

Nuclear magnetic resonance-based metabonomic study of early time point laminitis in an oligofructose-overload model.

REASONS FOR PERFORMING STUDY NMR-metabonomics is an unbiased evaluation method, which allows to comprehensively study changes of the equine metabolic profile in early time point laminitis. This might give insight into the early stages of disease development. OBJECTIVES To detect hitherto unknown changes in blood metabolites during the development of oligofructose-induced laminitis by comparing pre- and post induction blood samples. METHODS Prior to laminitis induction blood was sampled to establish control values. Post oligofructose administration (POA) blood was collected every 3 h for 24 h. One-dimensional (1) H-NMR spectra of the blood plasma were statistically analysed. RESULTS NMR resonances of >20 metabolites were identified using this technique. Already known changes (e.g. lactate concentrations) were confirmed using this method. Interestingly, oligofructose, a carbohydrate usually considered indigestible in the small intestine, or derivatives of oligofructose, was detected in plasma. Horses also showed increased phosphatidylcholine and/or low density lipoprotein levels POA, indicating a change in blood lipid composition. An increase in phosphatidylcholine is consistent with the breakdown of the mucosal layer of the large intestine and increased permeability of the gut. CONCLUSION Due to the nontargeted approach of metabonomics, new unexpected changes can be identified, in this case the hitherto unknown oligofructose uptake through the mucosal wall and the phospholipid changes. POTENTIAL RELEVANCE Metabolic changes in disease can be observed using NMR metabonomics. Oligofructose is used in feedstuffs and transport mechanisms through the mucosa should be studied. Phospholipids could point to a compromise of the intestinal wall during laminitis development.

Retrocyclin-2: a potent anti-HIV theta-defensin that forms a cyclic cystine ladder structural motif.

Retrocyclin-2: a potent anti-HIV theta-defensin that forms a cyclic cysteine ladder structural motif

Design and characterization of a soluble fragment of the extracellular ligand-binding domain of the peptide hormone receptor guanylyl cyclase-C

The intestinal guanylyl cyclase-C (GC-C) was originally identified as an Escherichia coli heat-stable enterotoxin (STa) receptor. STa stimulates GC-C to much higher activity than the endogenous ligands guanylin and uroguanylin, causing severe diarrhea. To investigate the interactions of the endogenous and bacterial ligands with GC-C, we designed and characterized a soluble and properly folded fragment of the extracellular ligand-binding domain of GC-C. The membrane-bound guanylyl cyclases exhibit a single transmembrane spanning helix and a globularly folded extracellular ligand-binding domain that comprises about 410 of 1050 residues. Based on the crystal structure of the dimerized-binding domain of the guanylyl cyclase-coupled atrial natriuretic peptide receptor and a secondary structure-guided sequence alignment, we generated a model of the extracellular domain of GC-C comprised of two subdomains. Mapping of mutational and cross-link data onto this structural model restricts the ligand-binding region to the membrane proximal subdomain. We thus designed miniGC-C, a 197 amino acid fragment that mimics the ligand-binding membrane proximal subdomain. Cloning, expression and spectroscopic studies reveal miniGC-C to be a soluble and properly folded protein with a distinct secondary and tertiary structure. MiniGC-C binds STa with nanomolar affinity.

Prosequence-Mediated Disulfide Coupled Folding of the Peptide Hormones Guanylin and Uroguanylin

In contrast to their prohormones the mature peptide hormones guanylin and uroguanylin are not able to fold to their native disulfide connectivities upon oxidative folding. Structural properties of both peptide hormones and their precursor proteins as well as the role of their prosequences in proper disulfide coupled folding are reviewed. In addition, the structural behavior of a proguanylin mutant that closely resembles prouroguanylin has been investigated to gain further insight into structural properties of this homologous precursor protein.