Characterisation of proteolytic enzymes of Eurygaster integriceps Put. (Sunn bug), a major pest of cereals

Eurygaster integriceps (Sunn pest or Sunn bug) is one of the most significant pests of wheat and is responsible for substantial losses in yield and quality of wheat grain in Europe and Asia. Sunn pest salivary gland-derived proteases and other hydrolases damage grain proteins and starch. Characterisation of protease activities from both Sunn pest salivary glands and Sunn pest-damaged wheat grains revealed a broad range of activities in terms of substrate specificity and diversity of isoelectric point. Neutral and alkaline proteases present in Sunn pest-damaged grains were shown to be capable of hydrolyzing gluten proteins, whilst some proteases were also shown to be active against gelatin. The neutral serine proteases present play the dominant role in degradation of gluten quality. The sensitivity of some proteases to proteinaceous and non-proteinaceous serine proteinase inhibitors was shown, including that of a recombinantly expressed protease. It was found that proteases isolated from Sunn pest salivary glands could be activated by trypsin indicating that they are present as zymogens in vivo. Analysis of individual Sunn pest-damaged grains showed great diversity in the proteases present. This work highlights the challenges of developing proteinase inhibitors to manage Sunn pest damage

Colloidal stability of tannins: astringency, wine tasting and beyond

Tannin-tannin and tannin-protein interactions in water-ethanol solvent mixtures are studied in the context of red wine tasting. While tannin self-aggregation is relevant for visual aspect of wine tasting (limpidity and related colloidal phenomena), tannin affinities for salivary proline-rich proteins is fundamental for a wide spectrum of organoleptic properties related to astringency. Tannin-tannin interactions are analyzed in water-ethanol wine-like solvents and the precipitation map is constructed for a typical grape tannin. The interaction between tannins and human salivary proline-rich proteins (PRP) are investigated in the framework of the shell model for micellization, known for describing tannin-induced aggregation of beta-casein. Tannin-assisted micellization and compaction of proteins observed by SAXS are described quantitatively and discussed in the case of astringency

(5,10,15,20-Tetra­phenyl­porphyrinato-κ4 N)cobalt(II)–18-crown-6 (1/1)

The asymmetric unit of the title compound, [Co(C44H28N4)]·C12H24O6, contains one half of a CoII(TPP) (TPP is tetra­phenyl­porphyrin) complex and one half of an 18-crown-6 mol­ecule of crystallization, both lying on inversion centers. The CoII(TPP) complex exhibits a nearly planar conformation of the porphyrinate core [maximum deviation = 0.069 (2) Å] with an average Co—N distance of 1.971 (4) Å. The distance between the Co atom and the closest O atom of the 18-crown-6 mol­ecule is 2.533 (2) Å, indicating a short non-bonded contact between the Co atom and the crown ether mol­ecule. An ethyl­ene group of the 18-crown-6 mol­ecule is disordered over two sites with occupancies of 0.565 (7) and 0.435 (7)

Active-Site-Directed Inhibitors of Prolyl Oligopeptidase Abolish Its Conformational Dynamics

Deciphering conformational dynamics is crucial for understanding the biological functions of proteins and for designing compounds targeting them. In particular, providing an accurate description of microsecond–millisecond motions opens the opportunity for regulating protein–protein interactions (PPIs) by modulating the dynamics of one interacting partner. Here we analyzed the conformational dynamics of prolyl oligopeptidase (POP) and the effects of active-site-directed inhibitors on the dynamics. We used an integrated structural biology approach based on NMR spectroscopy and SAXS experiments complemented by MD simulations. We found that POP is in a slow equilibrium in solution between open and closed conformations, and that inhibitors effectively abolished this equilibrium by stabilizing the enzyme in the closed conformation.This work was supported by the Institute for Research in Biomedicine, MINECO-FEDER (Bio2013-40716-R, CTQ2013-48287 and CTQ2012-32183/BQU), and the Generalitat de Catalunya (XRB and Grup Consolidat 2014SGR521). AL has received funding from the Instituto de Salud Carlos III. PB acknowledges the Agence Nationale de la Recherche (SPINHD-ANR-CHEX-2011) and the ATIP-Avenir program for financial support. FHT’s fellowship is co-funded by the INSERM and the University of Copenhagen. Technical assistance from staff at the P12 beam line (EMBL/DESY) is acknowledged.Peer ReviewedPostprint (author's final draft

Antibodies raised against a Sunn bug (Eurygaster integriceps Put.) recombinant protease, rGHP3p2, can inhibit gluten‐hydrolyzing activity

Sunn pest or Sunn bug, Eurygaster integriceps Put., salivary gland proteases are responsible for the deterioration of wheat flour quality during dough mixing, resulting from gluten hydrolysis. These proteases are highly heterogeneous and show low sensitivity to most types of proteinaceous inhibitors, meaning that such inhibitors cannot be used to prevent gluten damage. The present study describes the generation of a specific peptide antibody, raised against the active center of the recombinant gluten-hydrolyzing protease (GHP3). The recombinant protein, encoding two repeats of the GHP3 sequence element involved in forming the S4 pocket and binding of substrate at position P4, was designed and expressed in Escherichia coli. The antibodies raised to this recombinant protein showed inhibitory activity against the GHP3 protease. The results indicate that it is possible to design specific antibodies to inhibit wheat-bug gluten-hydrolyzing proteases

Poly-Ig tandems from I-band titin share extended domain arrangements irrespective of the distinct features of their modular constituents

The cellular function of the giant protein titin in striated muscle is a major focus of scientific attention. Particularly, its role in passive mechanics has been extensively investigated. In strong contrast, the structural details of this filament are very poorly understood. To date, only a handful of atomic models from single domain components have become available and data on poly-constructs are limited to scarce SAXS analyses. In this study, we examine the molecular parameters of poly-Ig tandems from I-band titin relevant to muscle elasticity. We revisit conservation patterns in domain and linker sequences of I-band modules and interpret these in the light of available atomic structures of Ig domains from muscle proteins. The emphasis is placed on features expected to affect inter-domain arrangements. We examine the overall conformation of a 6Ig fragment, I65-I70, from the skeletal I-band of soleus titin using SAXS and electron microscopy approaches. The possible effect of highly conserved glutamate groups at the linkers as well as the ionic strength of the medium on the overall molecular parameters of this sample is investigated. Our findings indicate that poly-Ig tandems from I-band titin tend to adopt extended arrangements with low or moderate intrinsic flexibility, independently of the specific features of linkers or component Ig domains across constitutively- and differentially-expressed tandems. Linkers do not appear to operate as free hinges so that lateral association of Ig domains must occur infrequently in samples in solution, even that inter-domain sequences of 4-5 residues length would well accommodate such geometry. It can be expected that this principle is generally applicable to all Ig-tandems from I-band titi

Photoinduced C70 radical anions in polymer:fullerene blends

Photoinduced polarons in solid films of polymer-fullerene blends were studied by photoluminescence (PL), photoinduced absorption (PIA) and electron spin resonance (ESR). The donor materials used were P3HT and MEH-PPV. As acceptors we employed PC60BM as reference and various soluble C70-derivates: PC70BM, two different diphenylmethano-[70]fullerene oligoether (C70-DPM-OE) and two dimers, C70-C70 and C60-C70. Blend films containing C70 revealed characteristic spectroscopic signatures not seen with C60. Light-induced ESR showed signals at g\geq2.005, assigned to an electron localized on the C70 cage. The formation of C70 radical anions also leads to a subgap PIA band at 0.92 eV, hidden in the spectra of C70-based P3HT and MEH-PPV blends, which allows for more exact studies of charge separated states in conjugated polymer:C70 blends.Comment: 3 pages, 4 figures, accepted for PSS RR

Glutamate 270 plays an essential role in K activation and domain closure of Thermus thermophilus isopropylmalate dehydrogenase

The mutant E270A of Thermus thermophilus 3-isopropylmalate dehydrogenase exhibits largely reduced (∼1%) catalytic activity and negligible activation by K+ compared to the wild-type enzyme. A 3–4 kcal/mol increase in the activation energy of the catalysed reaction upon this mutation could also be predicted by QM/MM calculations. In the X-ray structure of the E270A mutant a water molecule was observed to take the place of K+. SAXS and FRET experiments revealed the essential role of E270 in stabilisation of the active domain-closed conformation of the enzyme. In addition, E270 seems to position K+ into close proximity of the nicotinamide ring of NAD+ and the electron-withdrawing effect of K+ may help to polarise the aromatic ring in order to aid the hydride-transfer