Contact and channel modelling to support the early design of technical systems

The early design of mechanical systems is critical because it constrains options later in the design process. In this early stage of the design process, designers must consider customer requirements, how they are related to system functionality and how these requirements and functions are implemented in the physical interactions between components and sub-systems. This paper proposes an approach by which the Contact and Channel Model (C&CM) can be applied to support this stage of embodiment design. The proposed approach is implemented in a computer support tool and illustrated through a simple example. A number of opportunities for further work to extend and evaluate the approach are identified. We argue that our approach offers a unique way to consider the functionality of a design alongside the parts, surfaces and physical effects which embody that functionality, and provides an intuitive representation which could help designers iteratively develop and express this relationship

TransCent: Computational enzyme design by transferring active sites and considering constraints relevant for catalysis

BACKGROUND: Computational enzyme design is far from being applicable for the general case. Due to computational complexity and limited knowledge of the structure-function interplay, heuristic methods have to be used. RESULTS: We have developed TransCent, a computational enzyme design method supporting the transfer of active sites from one enzyme to an alternative scaffold. In an optimization process, it balances requirements originating from four constraints. These are 1) protein stability, 2) ligand binding, 3) pKa values of active site residues, and 4) structural features of the active site. Each constraint is handled by an individual software module. Modules processing the first three constraints are based on state-of-the-art concepts, i.e. RosettaDesign, DrugScore, and PROPKA. To account for the fourth constraint, knowledge-based potentials are utilized. The contribution of modules to the performance of TransCent was evaluated by means of a recapitulation test. The redesign of oxidoreductase cytochrome P450 was analyzed in detail. As a first application, we present and discuss models for the transfer of active sites in enzymes sharing the frequently encountered triosephosphate isomerase fold. CONCLUSION: A recapitulation test on native enzymes showed that TransCent proposes active sites that resemble the native enzyme more than those generated by RosettaDesign alone. Additional tests demonstrated that each module contributes to the overall performance in a statistically significant manner

Tiokarbamát-maradványok gáz - folyadék kromatográfiás meghatározása

Die Methode ist als schnelle und selektive gaschromatographische Bestimmung der Rückständen von fünf Thiokarbamat-Herbiziden (EPTC, Butilate, Pebulate, Molinate, Cycloate). Die angewandte Säulenfüllung war 3% OV - 17/Gas Chrom Q 150—180 /im, die Detektion erfolgte durch einen phosphor- und stickstoffselektiven Alkaliflammenionisationsdetektor. Die Peakfläche war im Beriech 0,01 — 100 ng mit guter Korrelation für alle untersuchten Tiokarbamaten linear. Zur Bestimmung nebeneinander der fünf Verbindungen ist ein Temperaturprogramm anzuwenden. Bei Mustern pflanzlicher Herkunft ist eine Nachweisgrenee + 0,005 mg/kg für EPTC zu erreichen. The method can be used for selective and quick GLC determination of the residues of five thiolcarbamate herbicides (EPTC, butilate, pebulate, molinate, Cycloate). Column packing: 3% OV—17 on Gas Chrom Q 150— 180 pm. The detection was carried out with phosphor and nitrogen selective alkali flame ionization detector (PN-AFID). The peak area proved to be linear with strict correlation in the range of 0.01 — 100 ng in case of all examined thiolcarbamate. For simultaneous determination of the compounds application of temperature programming is necessary. Detection limit is 0.005 mg/kg measuring EPTC in samples of plant origin. On emploie une méthode chromatographique vite et selective pour le dosage des résidues de cinq thiocarbamates-herbicides (EPTC, butylate, pebulate, molinate, cycloate). La colonne est chargée de 3% OV— 17 sur un support Gas Chrom Q 150-180 цт. La détection est réalisée avec un détecteur d’ionisation ä flamme alcaline sélectif pour l’azote et phosphore. L’aire au-dessous du poin de rebroussement a été constatée linéaire avec une bonne corrélation dans la région de 0,01 — 100 ng pour tous les thiocarbamates analysés. On doit pratiquer un programme ä temperature pour le dosage des composés les uns contre les autres. Cette méthode permet d’atteindre de sensibilité de detection de 0,005 mg/kg en cas du dosage de la teneur en EPTC des échantillons de plantes

Crystal structure of Saccharomyces cerevisiae mitochondrial GatFAB reveals a novel subunit assembly in tRNA-dependent amidotransferases

Yeast mitochondrial Gln-mtRNAGln is synthesized by the transamidation of mischarged Glu-mtRNAGln by a non-canonical heterotrimeric tRNA-dependent amidotransferase (AdT). The GatA and GatB subunits of the yeast AdT (GatFAB) are well conserved among bacteria and eukaryota, but the GatF subunit is a fungi-specific ortholog of the GatC subunit found in all other known heterotrimeric AdTs (GatCAB). Here we report the crystal structure of yeast mitochondrial GatFAB at 2.0 Å resolution. The C-terminal region of GatF encircles the GatA-GatB interface in the same manner as GatC, but the N-terminal extension domain (NTD) of GatF forms several additional hydrophobic and hydrophilic interactions with GatA. NTD-deletion mutants displayed growth defects, but retained the ability to respire. Truncation of the NTD in purified mutants reduced glutaminase and transamidase activities when glutamine was used as the ammonia donor, but increased transamidase activity relative to the full-length enzyme when the donor was ammonium chloride. Our structure-based functional analyses suggest the NTD is a trans-acting scaffolding peptide for the GatA glutaminase active site. The positive surface charge and novel fold of the GatF-GatA interface, shown in this first crystal structure of an organellar AdT, stand in contrast with the more conventional, negatively charged bacterial AdTs described previousl

The small GTPase Arf1 regulates ATP synthesis and mitochondria homeostasis by modulating fatty acid metabolism

Lipid mobilization through fatty acid β-oxidation is a central process essential for energy 36 production during nutrient shortage. In yeast, this catabolic process starts in the peroxisome from where β-oxidation products enter mitochondria and fuel the TCA cycle. Little is known about the physical and metabolic cooperation between these organelles. We found that expression of fatty acid transporters and of the rate-limiting enzyme involved in β-oxidation are decreased in cells expressing a hyperactive mutant of the small GTPase Arf1, leading to an accumulation of fatty acids in lipid droplets. As a consequence, mitochondria became fragmented and ATP synthesis decreased. Genetic and pharmacological depletion of fatty acids phenocopied the arf1 mutant mitochondrial phenotype. Although β-oxidation occurs mainly in mitochondria in mammals, Arf1's role in fatty acid metabolism is conserved. Together, our results indicate that Arf1 integrates metabolism into energy production by regulating fatty acid storage and utilization, and presumably organelle contact-sites

Nonconventional localizations of cytosolic aminoacyl-tRNA synthetases in yeast and human cells

International audienceKeywords: aaRS tRNA Yeast Human Microscopy Fractionation MTS NLS a b s t r a c t By definition, cytosolic aminoacyl-tRNA synthetases (aaRSs) should be restricted to the cytosol of eukary-otic cells where they supply translating ribosomes with their aminoacyl-tRNA substrates. However, it has been shown that other translationally-active compartments like mitochondria and plastids can simultaneously contain the cytosolic aaRS and its corresponding organellar ortholog suggesting that both forms do not share the same organellar function. In addition, a fair number of cytosolic aaRSs have also been found in the nucleus of cells from several species. Hence, these supposedly cytosolic-restricted enzymes have instead the potential to be multi-localized. As expected, in all examples that were studied so far, when the cytosolic aaRS is imported inside an organelle that already contains its bona fide corresponding organellar-restricted aaRSs, the cytosolic form was proven to exert a nonconventional and essential function. Some of these essential functions include regulating homeostasis and protecting against various stresses. It thus becomes critical to assess meticulously the subcellular localization of each of these cytosolic aaRSs to unravel their additional roles. With this objective in mind, we provide here a review on what is currently known about cytosolic aaRSs multi-compartmentalization and we describe all commonly used protocols and procedures for identifying the compartments in which cytosolic aaRSs relocal-ize in yeast and human cells