Crystal structure of Schmallenberg orthobunyavirus nucleoprotein-RNA complex reveals a novel RNA sequestration mechanism

Schmallenberg virus (SBV) is a newly emerged orthobunyavirus (family Bunyaviridae) that has caused severe disease in the offspring of farm animals across Europe. Like all orthobunyaviruses, SBV contains a tripartite negative-sense RNA genome that is encapsidated by the viral nucleocapsid (N) protein in the form of a ribonucleoprotein complex (RNP). We recently reported the three-dimensional structure of SBV N that revealed a novel fold. Here we report the crystal structure of the SBV N protein in complex with a 42-nt-long RNA to 2.16 Å resolution. The complex comprises a tetramer of N that encapsidates the RNA as a cross-shape inside the protein ring structure, with each protomer bound to 11 ribonucleotides. Eight bases are bound in the positively charged cleft between the N- and C-terminal domains of N, and three bases are shielded by the extended N-terminal arm. SBV N appears to sequester RNA using a different mechanism compared with the nucleoproteins of other negative-sense RNA viruses. Furthermore, the structure suggests that RNA binding results in conformational changes of some residues in the RNA-binding cleft and the N- and C-terminal arms. Our results provide new insights into the novel mechanism of RNA encapsidation by orthobunyaviruses

Protein disulfide-isomerase interacts with a substrate protein at all stages along its folding pathway

In contrast to molecular chaperones that couple protein folding to ATP hydrolysis, protein disulfide-isomerase (PDI) catalyzes protein folding coupled to formation of disulfide bonds (oxidative folding). However, we do not know how PDI distinguishes folded, partly-folded and unfolded protein substrates. As a model intermediate in an oxidative folding pathway, we prepared a two-disulfide mutant of basic pancreatic trypsin inhibitor (BPTI) and showed by NMR that it is partly-folded and highly dynamic. NMR studies show that it binds to PDI at the same site that binds peptide ligands, with rapid binding and dissociation kinetics; surface plasmon resonance shows its interaction with PDI has a Kd of ca. 10−5 M. For comparison, we characterized the interactions of PDI with native BPTI and fully-unfolded BPTI. Interestingly, PDI does bind native BPTI, but binding is quantitatively weaker than with partly-folded and unfolded BPTI. Hence PDI recognizes and binds substrates via permanently or transiently unfolded regions. This is the first study of PDI's interaction with a partly-folded protein, and the first to analyze this folding catalyst's changing interactions with substrates along an oxidative folding pathway. We have identified key features that make PDI an effective catalyst of oxidative protein folding – differential affinity, rapid ligand exchange and conformational flexibility

Effect of the Strawberry Genotype, Cultivation and Processing on the Fra a 1 Allergen Content

Birch pollen allergic patients show cross-reactivity to vegetables and fruits, including strawberries (Fragaria × ananassa). The objective of this study was to quantify the level of the Fra a 1 protein, a Bet v 1-homologous protein in strawberry fruits by a newly developed ELISA, and determine the effect of genotype, cultivation and food processing on the allergen amount. An indirect competitive ELISA using a specific polyclonal anti-Fra a 1.02 antibody was established and revealed high variability in Fra a 1 levels within 20 different genotypes ranging from 0.67 to 3.97 μg/g fresh weight. Mature fruits of red-, white- and yellow-fruited strawberry cultivars showed similar Fra a 1 concentrations. Compared to fresh strawberries, oven and solar-dried fruits contained slightly lower levels due to thermal treatment during processing. SDS-PAGE and Western blot analysis demonstrated degradation of recombinant Fra a 1.02 after prolonged (>10 min) thermal treatment at 99 ◦ C. In conclusion, the genotype strongly determined the Fra a 1 quantity in strawberries and the color of the mature fruits does not relate to the amount of the PR10-protein. Cultivation conditions (organic and conventional farming) do not affect the Fra a 1 level, and seasonal effects were minor

Matrix product state comparison of the numerical renormalization group and the variational formulation of the density matrix renormalization group

Wilson's numerical renormalization group (NRG) method for solving quantum impurity models yields a set of energy eigenstates that have the form of matrix product states (MPS). White's density matrix renormalization group (DMRG) for treating quantum lattice problems can likewise be reformulated in terms of MPS. Thus, the latter constitute a common algebraic structure for both approaches. We exploit this fact to compare the NRG approach for the single-impurity Anderson model to a variational matrix product state approach (VMPS), equivalent to single-site DMRG. For the latter, we use an ``unfolded'' Wilson chain, which brings about a significant reduction in numerical costs compared to those of NRG. We show that all NRG eigenstates (kept and discarded) can be reproduced using VMPS, and compare the difference in truncation criteria, sharp vs. smooth in energy space, of the two approaches. Finally, we demonstrate that NRG results can be improved upon systematically by performing a variational optimization in the space of variational matrix product states, using the states produced by NRG as input.Comment: 19 pages, 14 figure

Structural history of the southwestern corner of the Kaapvaal Craton and the adjacent Namaqua realm: new observations and a reappraisal

The rocks along the southwestern margin of the Kaapvaal Craton were deformed up to 7 times during the Early to Middle Proterozoic. The oldest deformation D1 is recorded in the N-S-trending Uitkoms cataclasite of pre-Makganyene age (>2.24 Ga) on the craton, and interpreted as a bedding-parallel thrust. It is assumed to be a branch rising towards the surface from a blind sole thrust that initiated early N-S-trending F,-folds above it. D2 is represented by mainly N-S but also NE-SW and NW-SE-trending imbricates and recumbent fold zones ranging in size from small gravity slumps to large tectonic decollements in Asbesheuwel BIF and the Koegas Subgroup, and is younger than D1, or equals D1 in age. These age. These structures pre-date the Westerberg dyke-sheet intrusion. D3 south-verging folds and thrusts are the oldest post-Matsap deformations, just less than 2.07-1.88 Ga. D4 are upright to east vergent and N-S-trending folds deforming all previous structures. D4 post-dates the Westerberg dyke-sheet and probably reactivates N-S folds above the earlier sole thrust during renewed E-W compression. D5, producing the main NW-trending Namaqua structures, is only very feebly developed in the Kheis terrain and absent from the cratonic areas overlain by Olifantshoek and older strata, i.e. NE, E and SE of the Marydale High. Very gentle D6 E-W to ENE-WSW folds produce culminations and depressions in all NW-trending older structures. During D7 the NW-SE-trending Doornberg Lineament, an oblique left-lateral wrench, and smaller N-trending faults such as the Westerberg Fault developed. These and similar, but right-lateral faults are the last movements along the rim of the craton and occurred around 1.0 Ga. Multiple folding and thrusting with riebeckite mobilization happened prior to Namaqua events and resulted inter alia in discernable duplication and thickening of the Transvaal Supergroup along the southwestern margin of the Kaapvaal Craton and at least some 130 km into the craton interior. This complicates stratigraphic correlation as well as true thickness estimates of BIF units in Griqualand West, and affects the model for the environmental evolution of the Ghaap Group. A structural model of thin-skin decoupling at the base of the Transvaal Supergroup and starting in the Middle-Early Proterozoic is proposed

Cloning, Expression, Sequence Analysis and Homology Modeling of the Prolyl Endoprotease from Eurygaster integriceps Puton

Eurygaster integriceps Puton, commonly known as sunn pest, is a major pest of wheat in Northern Africa, the Middle East and Eastern Europe. This insect injects a prolyl endoprotease into the wheat, destroying the gluten. The purpose of this study was to clone the full length cDNA of the sunn pest prolyl endoprotease (spPEP) for expression in E. coli and to compare the amino acid sequence of the enzyme to other known PEPs in both phylogeny and potential tertiary structure. Sequence analysis shows that the 5ꞌ UTR contains several putative transcription factor binding sites for transcription factors known to be expressed in Drosophila that might be useful targets for inhibition of the enzyme. The spPEP was first identified as a prolyl endoprotease by Darkoh et al., 2010. The enzyme is a unique serine protease of the S9A family by way of its substrate recognition of the gluten proteins, which are greater than 30 kD in size. At 51% maximum identity to known PEPs, homology modeling using SWISS-MODEL, the porcine brain PEP (PDB: 2XWD) was selected in the database of known PEP structures, resulting in a predicted tertiary structure 99% identical to the porcine brain PEP structure. A Km for the recombinant spPEP was determined to be 210 ± 53 µM for the zGly-Pro-pNA substrate in 0.025 M ethanolamine, pH 8.5, containing 0.1 M NaCl at 37 °C with a turnover rate of 172 ± 47 µM Gly-Pro-pNA/s/µM of enzyme

The refolding of recombinant human liver methylmalonyl-CoA mutase from inclusion bodies produced in Escherichia coli : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University

Human methylmalonyl-CoA mutase (hMCM) is an adenosylcobalamin-dependent enzyme that catalyses the structural rearrangement of (R)-methylmalonyl-CoA to succinyl-CoA as pan of the catabolism of the branched chain amino acids valine, leucine and isoleucine, odd chain fatty acids and intermediates of cholesterol metabolism. Reactions that require adenosylcobalamin (AdoCbl) have been intensively studied, and the first step in the catalysis is widely agreed to involve homolytic cleavage of the unusual carbon-cobalt bond in the cofactor. A reliable source of recombinant hMCM would be useful in defining more fully the mechanistic pathway of AdoCbl-dependent enzymes. Recombinant hMCM overexpressed in E. coli forms insoluble aggregates of inactive protein known as inclusion bodies. hMCM inclusion bodies were purified, solubilised and then several different in vitro refolding techniques were tested in attempts to produce active recombinant hMCM from purified solubilised inclusion body material. These methods included refolding by rapid dilution, refolding by dialysis, detergent-assisted refolding, refolding by gel filtration chromatography and chaperonin-assisted refolding. Chaperonin-assisted refolding necessitated the purification of recombinant E. coli chaperonins GroES and GroEL from the E. coli strain DH1/pGroESL. Refolding by rapid dilution of the GdmHCl-solubilised inclusion bodies into a refolding buffer was judged to be the simplest and most effective method, however the refolding process was extremely inefficient. Refolding by rapid dilution was scaled up to 2 litres to produce as much active hMCM as possible. The refolded protein was concentrated by batch adsorption to and stepwise elution from hydroxyapatite, and further purified using a synthesised 5'adenosylcobalamin- agarose 'affinity' chromatography column. The final refolded hMCM preparation contained a single ~29 kDa contaminant protein, tentatively identified as E. coli branched-chain amino acid aminotransferase (EC 2.6.1.42), present in approximately equal amounts to the hMCM, and had a specific activity of ~3.11 units/mg