An Arabidopsis thaliana arabinogalactan-protein (AGP31) and several cationic AGP fragments catalyse the boron bridging of rhamnogalacturonan-II

Rhamnogalacturonan-II (RG-II) is a complex pectic domain in plant primary cell walls. In vivo, most RG-II domains are covalently dimerised via borate diester bridges, essential for correct cell-wall assembly, but the dimerisation of pure RG-II monomers by boric acid in vitro is extremely slow. Cationic ‘chaperones’ can promote dimerisation, probably by overcoming the mutual repulsion between neighbouring anionic RG-II molecules. Highly effective artificial chaperones include Pb(2+) and polyhistidine, but the proposed natural chaperones remained elusive. We have now tested cationic peptide fragments of several Arabidopsis thaliana arabinogalactan-proteins (AGPs) as candidates. Fragments of AGP17, 18, 19 and 31 were effective, typically at ∼25 µg/ml (9–19 µM), promoting the boron bridging of 16–20 µM monomeric RG-II at pH 4.8 in vitro. Native AGP31 glycoprotein was also effective, and hexahistidine was moderately so. All chaperones tested interacted reversibly with RG-II and were not consumed during the reaction; thus they acted catalytically, and may constitute the first reported boron-acting enzyme activity, an RG-II borate diesterase. Many of the peptide chaperones became less effective catalysts at higher concentration, which we interpret as due to the formation of RG-II–peptide complexes with a net positive charge, as mutually repulsive as negatively charged pure RG-II molecules. The four unique AGPs studied here may serve an enzymic role in the living plant cell, acting on RG-II within Golgi cisternae and/or in the apoplast after secretion. In this way, RG-II and specific AGPs may contribute to cell-wall assembly and hence plant cell expansion and development

Outer membrane protein folding from an energy landscape perspective

The cell envelope is essential for the survival of Gram-negative bacteria. This specialised membrane is densely packed with outer membrane proteins (OMPs), which perform a variety of functions. How OMPs fold into this crowded environment remains an open question. Here, we review current knowledge about OFMP folding mechanisms in vitro and discuss how the need to fold to a stable native state has shaped their folding energy landscapes. We also highlight the role of chaperones and the β-barrel assembly machinery (BAM) in assisting OMP folding in vivo and discuss proposed mechanisms by which this fascinating machinery may catalyse OMP folding

A unified model for BAM function that takes into account type Vc secretion and species differences in BAM composition

Transmembrane proteins in the outer membrane of Gram-negative bacteria are almost exclusively β-barrels. They are inserted into the outer membrane by a conserved and essential protein complex called the BAM (for β-barrel assembly machinery). In this commentary, we summarize current research into the mechanism of this protein complex and how it relates to type V secretion. Type V secretion systems are autotransporters that all contain a β-barrel transmembrane domain inserted by BAM. In type Vc systems, this domain is a homotrimer. We argue that none of the current models are sufficient to explain BAM function particularly regarding type Vc secretion. We also find that current models based on the well-studied model system Escherichia coli mostly ignore the pronounced differences in BAM composition between different bacterial species. We propose a more holistic view on how all OMPs, including autotransporters, are incorporated into the lipid bilayer

On the thermal and thermomechanical assessment of the “Optimized Conservative” helium-cooled lithium lead breeding blanket concept for DEMO

Within the framework of EUROfusion R&D activities a research campaign has been performed at CEA-Saclay, in close collaboration with the University of Palermo, in order to investigate thermal and thermomechanical performances of the “Optimized Conservative” concept of DEMO Helium-Cooled Lithium Lead breeding blanket (HCLL). Attention has been paid to the HCLL outboard equatorial module (OEM) when subjected to the steady state nominal loading scenario. To this purpose three simplified 3D models, characterized by an increasing level of detail, have been set-up taking into account, firstly, a single radial-toroidal slice, then a basic module geometric unity composed by two adjacent slices and adding, lastly, the peripheral poloidal region. This latter 3D model has allowed the assessment of the Caps potential influence on the module thermal and thermomechanical behaviour. For each investigated 3D model, thermal and thermomechanical analyses have been performed and a stress linearization procedure has been carried out in order to verify the fulfilment of the criteria prescribed by the RCC-MRx 2015 code. The study has been performed adopting a numerical approach, based on the Finite Element Method (FEM), and adopting the Siemens NX v. 10.0 software in order to discretize the geometric domain, whereas thermal and thermomechanical calculations have been carried out using the Cast3 M 2015 FEM code. The obtained results, herewith reported and critically discussed, allow predicting a good thermal and mechanical behaviour of the “Optimized Conservative” concept of DEMO HCLL OEM, even if some small modifications to the module cooling scheme should be performed in order to avoid the insurgence of hotspots where temperature is slightly above the Eurofer limit temperature (550 °C). This will entail, from the mechanical point of view, a reduction of the secondary stress amount which is the main responsible of the failure in RCC-MRx criteria verification within First Wall-Side Wall bend region

Role of asparagine 1134 in glucosidic bond and transglycosylation specificity of reuteransucrase from Lactobacillus reuteri 121

Glucansucrases from lactic acid bacteria convert sucrose into various α-glucans that differ greatly with respect to the glucosidic bonds present (e.g. dextran, mutan, alternan and reuteran). This study aimed to identify the structural features of the reuteransucrase from Lactobacillus reuteri 121 (GTFA) that determine its reaction specificity. We here report a detailed mutational analysis of a conserved region immediately next to the catalytic Asp1133 (putative transition-state stabilizing) residue in GTFA. The data show that Asn1134 is the main determinant of glucosidic bond product specificity in this reuteransucrase. Furthermore, mutations at this position greatly influenced the hydrolysis/transglycosylation ratio. Changes in this amino acid expands the range of glucan and gluco-oligosaccharide products synthesized from sucrose by mutant GTFA enzymes.