A modified strategy for sequence specific assignment of protein NMR spectra based on amino acid type selective experiments

The determination of the three-dimensional structure of a protein or the study of protein–ligand interactions requires the assignment of all relevant nuclei as an initial step. This is nowadays almost exclusively performed using triple-resonance experiments. The conventional strategy utilizes one or more pairs of three dimensional spectra to obtain redundant information and thus reliable assignments. Here, a modified strategy for obtaining sequence specific assignments based on two dimensional amino acid type selective triple-resonance experiments is proposed. These experiments can be recorded with good resolution in a relatively short time. They provide very specific and redundant information, in particular on sequential connectivities, that drastically increases the ease and reliability of the assignment procedure, done either manually or in an automated fashion. The new strategy is demonstrated with the protein domain PB1 from yeast CDC24p. Dedicated to Rüdiger Winter ( 06.04.2004)     

16S rDNA phylogeny and distribution of lin genes in novel hexachlorocyclohexane-degrading Sphingomonas strains

Hexachlorocyclohexane (HCH) is a highly recalcitrant pesticide that persists in soils. Three novel HCH-degrading strains (DS2, DS2-2 and DS3-1) were isolated after enrichment from HCH-contaminated soil from Germany. These strains efficiently degraded the alpha-, gamma- and delta-isomers of HCH, while strain DS3-1 also degraded beta-HCH. Based on 16S rDNA analysis, strain DS3-1 was closely related to Sphingomonas taejonensis, while strains DS2 and DS2-2 were closely related to Sphingomonas flava and seven HCH-degrading strains recently isolated from HCH-contaminated Spanish soil. Hence, geographic origin of the strains was not reflected in their phylogenetic affiliation. Subsequently, lin genes involved in HCH degradation, virtually identical to those from Sphingomonas paucimobilis strains UT26 from Japan and B90A from India, were identified in strains DS3-1, DS2, DS2-2 and five of the strains from Spain. The conserved lin gene sequences and structural organization, as well as the close association with IS6100, suggest a shared lin gene origin and recent horizontal gene transfer among phylogenetically diverged Sphingomonas strains in remote geographic locations. The loss of the ability to degrade gamma-HCH was associated with the deletion of the linA gene, probably due to recombination involving IS6100 elements, of which several copies are located in the lin cluster region.     

Characterization of the oligomer structure of recombinant human mannan-binding lectin

Mannan-binding lectin (MBL) belongs to a family of proteins called the collectins, which show large differences in their ultrastructures. These differences are believed to be determined by different N-terminal disulfide-bonding patterns. So far only the bonding pattern of two of the simple forms (recombinant rat MBL-C and bovine CL-43) have been determined. Recombinant MBL expressed in human cells was purified, and the structure of the N-terminal region was determined. Preliminary results on human plasma-derived MBL revealed high similarity to the recombinant protein. Here we report the structure of the N-terminal part of recombinant human MBL and present a model to explain the oligomerization pattern. Using a strategy of consecutive enzymatic digestions and matrix-assisted laser desorption ionization mass spectrometry, we succeeded in identifying a number of disulfide-linked peptides from the N-terminal cysteine-rich region. Based on these building blocks, we propose a model that can explain the various oligomeric forms found in purified MBL preparations. Furthermore, the model was challenged by the production of cysteine to serine mutants of the three N-terminally situated cysteines. The oligomerization patterns of these mutants support the proposed model. The model indicates that the polypeptide dimer is the basic unit in the oligomerization.     

The solution structure of the SODD BAG domain reveals additional electrostatic interactions in the HSP70 complexes of SODD subfamily BAG domains

The solution structure of an N-terminally extended construct of the SODD BAG domain was determined by nuclear magnetic resonance spectroscopy. A homology model of the SODD-BAG/HSP70 complex reveals additional possible interactions that are specific for the SODD subfamily of BAG domains while the overall geometry of the complex remains the same. Relaxation rate measurements show that amino acids N358-S375 of SODD which were previously assigned to its BAG domain are not structured in our construct. The SODD BAG domain is thus indeed smaller than the homologous domain in Bag1 defining a new subfamily of BAG domains.     

Crystal structure of the 270 kDa homotetrameric lignin-degrading enzyme pyranose 2-oxidase

Pyranose 2-oxidase (P2Ox) is a 270 kDa homotetramer localized preferentially in the hyphal periplasmic space of lignocellulolytic fungi and has a proposed role in lignocellulose degradation to produce the essential co-substrate, hydrogen peroxide, for lignin peroxidases. P2Ox oxidizes D-glucose and other aldopyranoses regioselectively at C2 to the corresponding 2-keto sugars; however, for some substrates, the enzyme also displays specificity for oxidation at C3. The crystal structure of P2Ox from Trametes multicolor has been determined using single anomalous dispersion with mercury as anomalous scatterer. The model was refined at 1.8A resolution to R and Rfree values of 0.134 and 0.171, respectively. The overall fold of the P2Ox subunit resembles that of members of the glucose-methanol-choline family of long-chain oxidoreductases, featuring a flavin-binding Rossmann domain of class alpha/beta and a substrate-binding subdomain with a six-stranded central beta sheet and three alpha helices. The homotetramer buries a large internal cavity of roughly 15,000 A3, from which the four active sites are accessible. Four solvent channels lead from the surface into the cavity through which substrate must enter before accessing the active site. The present structure shows an acetate molecule bound in the active site with the carboxylate group positioned immediately below the flavin N5 atom, and with one carboxylate oxygen atom interacting with the catalytic residues His548 and Asn593. The entrance to the active site is blocked by a loop (residues 452 to 461) with excellent electron density but elevated temperature factors. We predict that this loop is dynamic and opens to allow substrate entry and exit. In silico docking of D-glucose in the P2Ox active site shows that with the active-site loop in the closed conformation, monosaccharides cannot be accommodated; however, after removing the loop from the model, a tentative set of protein-substrate interactions for beta-D-glucose have been outlined.     

Proteomic identification of processes and pathways characteristic of osmoregulatory tissues in spiny dogfish shark (Squalus acanthias)

We used dogfish shark (Squalus acanthias) as a model for proteome analysis of six different tissues to evaluate tissue-specific protein expression on a global scale and to deduce specific functions and the relatedness of multiple tissues from their proteomes. Proteomes of heart, brain, kidney, intestine, gill, and rectal gland were separated by two-dimensional gel electrophoresis (2DGE), gel images were matched using Delta 2D software and then evaluated for tissue-specific proteins. Sixty-one proteins (4%) were found to be in only a single type of tissue and 535 proteins (36%) were equally abundant in all six tissues. Relatedness between tissues was assessed based on tissue-specific expression patterns of all 1465 consistently resolved protein spots. This analysis revealed that tissues with osmoregulatory function (kidney, intestine, gill, rectal gland) were more similar in their overall proteomes than non-osmoregulatory tissues (heart, brain). Sixty-one proteins were identified by MALDI-TOF/TOF mass spectrometry and biological functions characteristic of osmoregulatory tissues were derived from gene ontology and molecular pathway analysis. Our data demonstrate that the molecular machinery for energy and urea metabolism and the Rho-GTPase/cytoskeleton pathway are enriched in osmoregulatory tissues of sharks. Our work provides a strong rationale for further study of the contribution of these mechanisms to the osmoregulation of marine sharks.     

Synthesis and characterization of β-diketiminate germanium(II) and tin(II) bromides

The equimolar reaction of a β-diketiminate lithium salt LLi(OEt₂) [L = HC(CMeNAr)₂; Ar = 2,6-iPr₂C₆H₃] with either GeBr₂ or SnBr₂ in diethyl ether affords the synthetically useful monomeric β-diketiminate-element halides LGeBr (1) and LSnBr (2), respectively. Both are soluble in hydrocarbon solvents, stable in inert atmosphere, and have been characterized by elemental analysis, NMR spectroscopy, and single-crystal X-ray diffraction analysis.     

Differential regulation by organic compounds and heavy metals of multiple laccase genes in the aquatic hyphomycete Clavariopsis aquatica

To advance the understanding of the molecular mechanisms controlling microbial activities involved in carbon cycling and mitigation of environmental pollution in freshwaters, the influence of heavy metals and natural as well as xenobiotic organic compounds on laccase gene expression was quantified using quantitative real-time PCR (qRT-PCR) in an exclusively aquatic fungus (the aquatic hyphomycete Clavariopsis aquatica) for the first time. Five putative laccase genes (lcc1 to lcc5) identified in C. aquatica were differentially expressed in response to the fungal growth stage and potential laccase inducers, with certain genes being upregulated by, e.g., the lignocellulose breakdown product vanillic acid, the endocrine disruptor technical nonylphenol, manganese, and zinc. lcc4 is inducible by vanillic acid and most likely encodes an extracellular laccase already excreted during the trophophase of the organism, suggesting a function during fungal substrate colonization. Surprisingly, unlike many laccases of terrestrial fungi, none of the C. aquatica laccase genes was found to be upregulated by copper. However, copper strongly increases extracellular laccase activity in C. aquatica, possibly due to stabilization of the copper-containing catalytic center of the enzyme. Copper was found to half-saturate laccase activity already at about 1.8 μM, in favor of a fungal adaptation to low copper concentrations of aquatic habitats.