Conformational Transitions Accompanying Oligomerization of Yeast Alcohol Oxidase, a Peroxisomal Flavoenzyme

Alcohol oxidase (AO) is a homo-octameric flavoenzyme which catalyzes methanol oxidation in methylotrophic yeasts. AO protein is synthesized in the cytosol and subsequently sorted to peroxisomes where the active enzyme is formed. To gain further insight in the molecular mechanisms involved in AO activation, we studied spectroscopically native AO from Hansenula polymorpha and Pichia pastoris and three putative assembly intermediates. Fluorescence studies revealed that both Trp and FAD are suitable intramolecular markers of the conformation and oligomeric state of AO. A direct relationship between dissociation of AO octamers and increase in Trp fluorescence quantum yield and average fluorescence lifetime was found. The time-resolved fluorescence of the FAD cofactor showed a rapid decay component which reflects dynamic quenching due to the presence of aromatic amino acids in the FAD-binding pocket. The analysis of FAD fluorescence lifetime profiles showed a remarkable resemblance of pattern for purified AO and AO present in intact yeast cells. Native AO contains a high content of ordered secondary structure which was reduced upon FAD-removal. Dissociation of octamers into monomers resulted in a conversion of β-sheets into α-helices. Our results are explained in relation to a 3D model of AO, which was built based on the crystallographic data of the homologous enzyme glucose oxidase from Aspergillus niger. The implications of our results for the current model of the in vivo AO assembly pathway are discussed.

GPCR-OKB: the G protein coupled receptor oligomer knowledge base

Rapid expansion of available data about G Protein Coupled Receptor (GPCR) dimers/oligomers over the past few years requires an effective system to organize this information electronically. Based on an ontology derived from a community dialog involving colleagues using experimental and computational methodologies, we developed the GPCR-Oligomerization Knowledge Base (GPCR-OKB). GPCR-OKB is a system that supports browsing and searching for GPCR oligomer data. Such data were manually derived from the literature. While focused on GPCR oligomers, GPCR-OKB is seamlessly connected to GPCRDB, facilitating the correlation of information about GPCR protomers and oligomers

Термопружний стан шаруватих термочутливих циліндрів і куль за конвективно-променевого теплообміну

Розроблено методику розв’язування одновимірних статичних задач термопружності для шаруватих циліндрів і куль за конвективно-променевого нагрівання з урахуванням лінійної залежності коефіцієнтів теплопровідності та довільного характеру залежності інших фізико-механічних характеристик від температури. Задачі теплопровідності зведено, незалежно від кількості шарів, до розв’язування системи двох нелінійних алгебричних рівнянь. У задачах термопружності неперервні в межах кожного шару коефіцієнти рівнянь апроксимовано кусково-сталими функціями. Проведено числовий аналіз температурних полів і напружень у двошарових тілах.The method for solving one-dimensional static thermoelasticity problems is developed for layered cylinders and spheres under convective and radiation heating, taking into account linear dependence of heat conductivity coefficients and arbitrary character of dependence of other physical and mechanical characteristics on a temperature. Problems of heat conductivity have been reduced to the system of two nonlinear algebraic equations regardless of the number of layers. In the thermoelasticity problems the continuous within every layer coefficients of equations are approximated by piecewise constant functions. The numerical analysis of the temperature fields and stresses is conducted for two-layered bodies.Разработана методика решения одномерных статических задач термоупругости для слоистых цилиндров и сфер при конвективно-лучевом нагреве с учетом линейной зависимости коэффициентов теплопроводности и произвольного характера зависимости других физико-механических характеристик от температуры. Задачи теплопроводности сведены, независимо от количества слоев, к решению системы двух нелинейных алгебраических уравнений. В задачах термоупругости непрерывные в пределах каждого слоя коэффициенты уравнений аппроксимированы кусочно-постоянными функциями. Проведен численный анализ температурных полей и напряжений в двухслойных телах

Target and (Astro-)WISE technologies - Data federations and its applications

After its first implementation in 2003 the Astro-WISE technology has been rolled out in several European countries and is used for the production of the KiDS survey data. In the multi-disciplinary Target initiative this technology, nicknamed WISE technology, has been further applied to a large number of projects. Here, we highlight the data handling of other astronomical applications, such as VLT-MUSE and LOFAR, together with some non-astronomical applications such as the medical projects Lifelines and GLIMPS, the MONK handwritten text recognition system, and business applications, by amongst others, the Target Holding. We describe some of the most important lessons learned and describe the application of the data-centric WISE type of approach to the Science Ground Segment of the Euclid satellite.Comment: 9 pages, 5 figures, Proceedngs IAU Symposium No 325 Astroinformatics 201

Astro-WISE: Chaining to the Universe

The recent explosion of recorded digital data and its processed derivatives threatens to overwhelm researchers when analysing their experimental data or when looking up data items in archives and file systems. While current hardware developments allow to acquire, process and store 100s of terabytes of data at the cost of a modern sports car, the software systems to handle these data are lagging behind. This general problem is recognized and addressed by various scientific communities, e.g., DATAGRID/EGEE federates compute and storage power over the high-energy physical community, while the astronomical community is building an Internet geared Virtual Observatory, connecting archival data. These large projects either focus on a specific distribution aspect or aim to connect many sub-communities and have a relatively long trajectory for setting standards and a common layer. Here, we report "first light" of a very different solution to the problem initiated by a smaller astronomical IT community. It provides the abstract "scientific information layer" which integrates distributed scientific analysis with distributed processing and federated archiving and publishing. By designing new abstractions and mixing in old ones, a Science Information System with fully scalable cornerstones has been achieved, transforming data systems into knowledge systems. This break-through is facilitated by the full end-to-end linking of all dependent data items, which allows full backward chaining from the observer/researcher to the experiment. Key is the notion that information is intrinsic in nature and thus is the data acquired by a scientific experiment. The new abstraction is that software systems guide the user to that intrinsic information by forcing full backward and forward chaining in the data modelling.Comment: To be published in ADASS XVI ASP Conference Series, 2006, R. Shaw, F. Hill and D. Bell, ed

Распределение металлической фазы в синтетических алмазах типа «карбонадо»

Studying of a structural structure synthetic «carbonado» is spent by a method of electronic scanning microscopy. It is established, that the surface «carbonado» is covered by the large diamond crystals reaching 200 microns, having obviously expressed structure of growth. On a surface of these crystals observed drops of metal-catalyst (nickel) of a submicronic range. It is established, that the structure synthetic «carbonado» represents interpenetrating skeletons from diamond and ceramic-metal phases

The effect of cavity-filling mutations on the thermostability of Bacillus stearothermophilus neutral protease

Cavities in the hydrophobic core of the neutral protease of Bacillus stearothermophilus were analyzed using a three-dimensional model that was inferred from the crystal structure of thermolysin, the highly homologous neutral protease of B.thermoproteolyticus (85% sequence identity). Site-directed mutagenesis was used to fill some of these cavities, thereby improving hydrophobic packing in the protein interior. The mutations had small effects on the thermostability, even after drastic changes, such as Leu284 --> Trp and Met168 --> Trp. The effects on T50, the temperature at which 50% of the enzyme is irreversibly inactivated in 30 min, ranged from 0.0 to +0.4-degrees-C. These results can be explained by assuming that the mutations have positive and negative structural effects of approximately the same magnitude. Alternatively, it could be envisaged that the local unfolding steps, which render the enzyme susceptible towards autolysis and which are rate limiting in the process of thermal inactivation, are only slightly affected by alterations in the hydrophobic core

UV-induced ligand exchange in MHC class I protein crystals

High-throughput structure determination of protein−ligand complexes is central in drug development and structural proteomics. To facilitate such high-throughput structure determination we designed an induced replacement strategy. Crystals of a protein complex bound to a photosensitive ligand are exposed to UV light, inducing the departure of the bound ligand, allowing a new ligand to soak in. We exemplify the approach for a class of protein complexes that is especially recalcitrant to high-throughput strategies: the MHC class I proteins. We developed a UV-sensitive, “conditional”, peptide ligand whose UV-induced cleavage in the crystals leads to the exchange of the low-affinity lytic fragments for full-length peptides introduced in the crystallant solution. This “in crystallo” exchange is monitored by the loss of seleno-methionine anomalous diffraction signal of the conditional peptide compared to the signal of labeled MHC β2m subunit. This method has the potential to facilitate high-throughput crystallography in various protein families

On the complexity of Engh and Huber refinement restraints: the angle τ as example

The angle τ (backbone N—Cα—C) is the most contested Engh and Huber refinement target parameter. It is shown that this parameter is ‘correct’ as a PDB-wide average, but can be improved by taking into account residue types, secondary structures and many other aspects of our knowledge of the biophysical relations between residue type and protein structure