SAMPLEX: Automatic mapping of perturbed and unperturbed regions of proteins and complexes

<p>Abstract</p> <p>Background</p> <p>The activity of proteins within the cell is characterized by their motions, flexibility, interactions or even the particularly intriguing case of partially unfolded states. In the last two cases, a part of the protein is affected either by binding or unfolding and the detection of the respective perturbed and unperturbed region(s) is a fundamental part of the structural characterization of these states. This can be achieved by comparing experimental data of the same protein in two different states (bound/unbound, folded/unfolded). For instance, measurements of chemical shift perturbations (CSPs) from NMR ¹H-¹⁵N HSQC experiments gives an excellent opportunity to discriminate both moieties.</p> <p>Results</p> <p>We describe an innovative, automatic and unbiased method to distinguish perturbed and unperturbed regions in a protein existing in two distinct states (folded/partially unfolded, bound/unbound). The SAMPLEX program takes as input a set of data and the corresponding three-dimensional structure and returns the confidence for each residue to be in a perturbed or unperturbed state. Its performance is demonstrated for different applications including the prediction of disordered regions in partially unfolded proteins and of interacting regions in protein complexes.</p> <p>Conclusions</p> <p>The proposed approach is suitable for partially unfolded states of proteins, local perturbations due to small ligands and protein-protein interfaces. The method is not restricted to NMR data, but is generic and can be applied to a wide variety of information.</p

Российская интеллигенция: блеск и нищета духа

Hairy and enhancer of split 1, one of the main downstream effectors in Notch signaling, is a transcriptional repressor of the basic helix-loop-helix (bHLH) family. Using nuclear magnetic resonance methods, we have determined the structure and dynamics of a recombinant protein, H1H, which includes an N-terminal segment, b1, containing functionally important phosphorylation sites, the basic region b2, required for binding to DNA, and the HLH domain. We show that a proline residue in the sequence divides the protein in two parts, a flexible and disordered N-terminal region including b1 and a structured, mainly helical region comprising b2 and the HLH domain. Binding of H1H to a double strand DNA oligonucleotide was monitored through the chemical shift perturbation of backbone amide resonances, and showed that the interaction surface involves not only the b2 segment but also several residues in the b1 and HLH regions

Introduction to a special issue of Magnetic Resonance in honour of Robert Kaptein at the occasion of his 80th birthday

This publication, in honour of Robert Kaptein's 80th birthday, contains contributions from colleagues, many of whom have worked with him, and others who admire his work and have been stimulated by his research. The contributions show current research in biomolecular NMR, spin hyperpolarisation and spin chemistry, including CIDNP (chemically induced dynamic nuclear polarisation), topics to which he has contributed enormously. His proposal of the radical pair mechanism was the birth of the field of spin chemistry, and the laser CIDNP NMR experiment on a protein was a major breakthrough in hyperpolarisation research. He set milestones for biomolecular NMR by developing computational methods for protein structure determination, including restrained molecular dynamics and 3D NMR methodology. With a lac repressor headpiece, he determined one of the first protein structures determined by NMR. His studies of the lac repressor provided the first examples of detailed studies of protein nucleic acid complexes by NMR. This deepened our understanding of protein DNA recognition and led to a molecular model for protein sliding along the DNA. Furthermore, he played a leading role in establishing the cluster of NMR large-scale facilities in Europe. This editorial gives an introduction to the publication and is followed by a biography describing his contributions to magnetic resonance

Information-driven protein–DNA docking using HADDOCK: it is a matter of flexibility

Intrinsic flexibility of DNA has hampered the development of efficient protein−DNA docking methods. In this study we extend HADDOCK (High Ambiguity Driven DOCKing) [C. Dominguez, R. Boelens and A. M. J. J. Bonvin (2003) J. Am. Chem. Soc. 125, 1731–1737] to explicitly deal with DNA flexibility. HADDOCK uses non-structural experimental data to drive the docking during a rigid-body energy minimization, and semi-flexible and water refinement stages. The latter allow for flexibility of all DNA nucleotides and the residues of the protein at the predicted interface. We evaluated our approach on the monomeric repressor−DNA complexes formed by bacteriophage 434 Cro, the Escherichia coli Lac headpiece and bacteriophage P22 Arc. Starting from unbound proteins and canonical B-DNA we correctly predict the correct spatial disposition of the complexes and the specific conformation of the DNA in the published complexes. This information is subsequently used to generate a library of pre-bent and twisted DNA structures that served as input for a second docking round. The resulting top ranking solutions exhibit high similarity to the published complexes in terms of root mean square deviations, intermolecular contacts and DNA conformation. Our two-stage docking method is thus able to successfully predict protein−DNA complexes from unbound constituents using non-structural experimental data to drive the docking

Алгоритм проведення попередньої дегазації методом гравітаційного знещільнення вуглепородного масиву

Приводится алгоритм проведения предварительной дегазации при применении нового способа гравитационной разгрузки на угольных месторождениях Донбасса и Львовско-Волынского бассейнов. Среди главных эффектов, которые будут получены при внедрении способа – обеспечение безопасности работ на шахтах и промышленная добыча метана.The algorithm of realization of pre-degassing with using new way of gravitational unloading on coal fields of Donbas and Lviv-Volyn basins is given in this article. Guaranteeing of safety works in mines and industrial methane-mining are between main effects which will be got on conditions of application of this method

High-level expression of biologically active glycoprotein hormones in Pichia pastoris strains—selection of strain GS115, and not X-33, for the production of biologically active N-glycosylated 15N-labeled phCG

The methylotrophic yeast Pichia pastoris is widely used for the production of recombinant glycoproteins. With the aim to generate biologically active 15N-labeled glycohormones for conformational studies focused on the unravelling of the NMR structures in solution, the P. pastoris strains GS115 and X-33 were explored for the expression of human chorionic gonadotropin (phCG) and human follicle-stimulating hormone (phFSH). In agreement with recent investigations on the N-glycosylation of phCG, produced in P. pastoris GS115, using ammonia/glycerol-methanol as nitrogen/carbon sources, the N-glycosylation pattern of phCG, synthesized using NH4Cl/glucose–glycerol–methanol, comprised neutral and charged, phosphorylated high-mannose-type N-glycans (Man8–15GlcNAc2). However, the changed culturing protocol led to much higher amounts of glycoprotein material, which is of importance for an economical realistic approach of the aimed NMR research. In the context of these studies, attention was also paid to the site specific N-glycosylation in phCG produced in P. pastoris GS115. In contrast to the rather simple N-glycosylation pattern of phCG expressed in the GS115 strain, phCG and phFSH expressed in the X-33 strain revealed, besides neutral high-mannose-type N-glycans, also high concentrations of neutral hypermannose-type N-glycans (Manup-to-30GlcNAc2). The latter finding made the X-33 strain not very suitable for generating 15N-labeled material. Therefore, 15N-phCG was expressed in the GS115 strain using the new optimized protocol. The 15N-enrichment was evaluated by 15N-HSQC NMR spectroscopy and GLC-EI/MS. Circular dichroism studies indicated that 15N-phCG/GS115 had the same folding as urinary hCG. Furthermore, 15N-phCG/GS115 was found to be similar to the unlabeled protein in every respect as judged by radioimmunoassay, radioreceptor assays, and in vitro bioassays

Identification of a diagnostic structural motif reveals a new reaction intermediate and condensation pathway in kraft lignin formation

The authors gratefully acknowledge financial support of NWO, the Smart Mix Program of the Netherlands Ministry of Economic Affairs and the Netherlands Ministry of Education, Culture and Science. The NWO Large grant 175.107.301.10 is also gratefully acknowledged.Kraft lignin, the main by-product of the pulping industry, is an abundant, yet highly underutilized renewable aromatic polymer. During kraft pulping, the lignin undergoes extensive structural modification, with many labile native bonds being replaced by new, more recalcitrant ones. Currently little is known about the nature of those bonds and linkages in kraft lignin, information that is essential for its efficient valorization to renewable fuels, materials or chemicals. Here, we provide detailed new insights into the structure of softwood kraft lignin, identifying and quantifying the major native as well as kraft pulping-derived units as a function of molecular weight. De novo synthetic kraft lignins, generated from (isotope labelled) dimeric and advanced polymeric models, provided key mechanistic understanding of kraft lignin formation, revealing different process dependent reaction pathways to be operating. The discovery of a novel kraft-derived lactone condensation product proved diagnostic for the identification of a previously unknown homovanillin based condensation pathway. The lactone marker is found in various different soft- and hardwood kraft lignins, suggesting the general pertinence of this new condensation mechanism for kraft pulping. These novel structural and mechanistic insights will aid the development of future biomass and lignin valorization technologies.Publisher PDFPeer reviewe

A comprehensive framework of E2–RING E3 interactions of the human ubiquitin–proteasome system

Covalent attachment of ubiquitin to substrates is crucial to protein degradation, transcription regulation and cell signalling. Highly specific interactions between ubiquitin-conjugating enzymes (E2) and ubiquitin protein E3 ligases fulfil essential roles in this process. We performed a global yeast-two hybrid screen to study the specificity of interactions between catalytic domains of the 35 human E2s with 250 RING-type E3s. Our analysis showed over 300 high-quality interactions, uncovering a large fraction of new E2–E3 pairs. Both within the E2 and the E3 cohorts, several members were identified that are more versatile in their interaction behaviour than others. We also found that the physical interactions of our screen compare well with reported functional E2–E3 pairs in in vitro ubiquitination experiments. For validation we confirmed the interaction of several versatile E2s with E3s in in vitro protein interaction assays and we used mutagenesis to alter the E3 interactions of the E2 specific for K63 linkages, UBE2N(Ubc13), towards the K48-specific UBE2D2(UbcH5B). Our data provide a detailed, genome-wide overview of binary E2–E3 interactions of the human ubiquitination system

NMR resonance assignments of NarE, a putative ADP-ribosylating toxin from Neisseria meningitidis

NarE is a 16 kDa protein identified from Neisseria meningitidis, one of the bacterial pathogens responsible for meningitis. NarE belongs to the ADP-ribosyltransferase family and catalyses the transfer of ADP-ribose moieties to arginine residues in target protein acceptors. Many pathogenic bacteria utilize ADP-ribosylating toxins to modify and alter essential functions of eukaryotic cells. NarE was proposed to bind iron through a Fe–S center which is supposed to be implied in catalysis. We have produced and purified uniformly labeled 15N- and 15N/13C-NarE and assigned backbone and side-chain resonances using multidimensional heteronuclear NMR spectroscopy. These assignments provide the starting point for the three-dimensional structure determination of NarE and the characterization of the role of the Fe–S center in the catalytic mechanism

Увічнення пам’яті героїв Східної (Кримської) війни (1853 – 1856 рр.) у кінофотодокументах

Дана стаття розглядає увічнення пам’яті героїв Східної (Кримської) війни 1853–1856 рр. та святкування її 100-річчя в кінофотодокументах.This article is dedicated to perpetuation of the memory of the Eastern (Crimean) War heroes. It also shows the celebration of 100-year anniversary by means of the cine- and photo documents