NMR-gestützte Untersuchung der strukturellen Grundlage für die Signaltransduktion in der angeborenen Immunabwehr durch autokatalytische Änderungen von MAVS

Upon virus infection, the innate immune response can be triggered by RIG-I like receptors (RLR) recognizing viral RNA. As a result, the two caspase activation and recruitment domains (CARD)s of RLRs become exposed and activate the CARD of the mitochondrial antiviral signaling protein (MAVS) to form high molecular weight assemblies, which serve as a 'platform' for the activation of downstream signaling components. The N-terminal CARD of MAVS is responsible for this aggregation. Recombinant MAVS CARD forms fibers, which can trigger the conversion of endogenous inactive full-length MAVS into active aggregates. The mechanism of MAVS CARD mediated self-assembly is still poorly understood. In this thesis, the structure of the MAVS CARD monomer was determined at pH 3.0 by solution NMR. It adopts a typical Greek key bundle of six anti-parallel helices. Analysis of the backbone dynamics revealed the MAVS CARD is extremely rigid, with minor exceptions at the termini. Pull-down assays and NMR titration assays of the RIG-I CARDs with monomeric MAVS CARD mutants provided first insights into the interaction interface between these two proteins. Moreover, solid state NMR was employed to determine the structure of MAVS CARD in its fibrillar conformation. Only minor structural changes were observed relative to the monomeric state. Thus, MAVS activation is one of the first examples of a prion-like conversion from a monomeric to an aggregated state that does not require a substantial conformational change. The quaternary structure of MAVS CARD was determined by combining intermolecular NMR constraints with helical symmetry information from electron microscopy. Three different interaction surfaces were identified between the MAVS CARD domains within the filament. These results presented here will be of significant value for future studies on MAVS activity and regulation, and the role of higher-order assemblies in signal transduction.Virale RNA wird von Rezeptoren der Rig-I Familie (RLR) erkannt und löst so eine angeborene Immunantwort aus. Die RLRs setzen ihre beiden Caspase-Aktivierungs- und Rekrutierungsdomänen (CARD) frei und aktivieren die CARD des mitochondrialen antiviralen Signalproteins (MAVS). Dieses bildet so hochmolekulare Strukturen, die als Plattform für die Aktivierung weiterer Signaltransduktionsmoleküle dienen. Die N-terminale MAVS CARD ist für diese Aggregation ausreichend. Recombinante MAVS CARD bildet Fasern, die eine Konversion von endogenem, inaktivem Volllängern-MAVS zu aktiven Aggregaten auslösen. Der Mechanismus dieser Selbstassemblierung ist noch schlecht verstanden. In der vorliegenden Arbeit wurde die Struktur des MAVS CARD Monomers bei pH 3.0 durch Lösungs-NMR bestimmt. Es bildet ein typisches “Greek Key”-Bündel aus sechs antiparallelen Helices. Die Protein-Rückgrat-Dynamik zeigte, das MAVS CARD sehr rigide ist, mit Ausnahme der Termini. Pulldown- und NMR-Tirationsexperimente zur Bindung der RIG-I CARD an monomere MAVS CARD Mutanten haben erste Einblicke in die Interaktionsflächen zwischen den beiden Proteinen gegeben. Mithilfe von Festkörper-NMR wurde die Struktur von MAVS CARD Fasern bestimmt und gezeigt, dass nur geringfügige Strukturänderungen relativ zum Monomer auftreten. Daher stellt die Aktivierung von MAVS eines der ersten Beispiele einer Prion-ähnlichen Konversion von einem monomeren zu einem aggregierten Zustand dar, der keine substantiellen konformationellen Änderungen beinhaltet. Um die Quartärstruktur von MAVS CARD zu lösen, wurden intermolekulare NMR Distanzinformationen mit Negativkontrast-Elektronenmikroskopie und Röntgenfaserbeugung zur Bestimmung der helikalen Symmetrie kombiniert. Es konnten drei Kontaktflächen der MAVS CARD Protomere untereinander identifiziert werden. Diese Ergebnisse werden für zukünftige Studien zur Aktivierung und Regulation von MAVS, sowie zur Rolle von hochgeordneten Aggregaten in der Signaltransduktion von hohem Wert sein

A molecular mechanism of chaperone–client recognition

Molecular chaperones are essential in aiding client proteins to fold into their native structure and in maintaining cellular protein homeostasis. However, mechanistic aspects of chaperone function are still not well understood at the atomic level. We use nuclear magnetic resonance spectroscopy to elucidate the mechanism underlying client recognition by the adenosine triphosphate-independent chaperone Spy at the atomic level and derive a structural model for the chaperone-client complex. Spy interacts with its partially folded client Im7 by selective recognition of flexible, locally frustrated regions in a dynamic fashion. The interaction with Spy destabilizes a partially folded client but spatially compacts an unfolded client conformational ensemble. By increasing client backbone dynamics, the chaperone facilitates the search for the native structure. A comparison of the interaction of Im7 with two other chaperones suggests that the underlying principle of recognizing frustrated segments is of a fundamental nature

The Nitrogen Budget of Coastal Eastern Guangdong in the Last 15 Years

Nitrogen pollution has caused severe ecological and environmental crisis, especially in densely populated coastal regions. Using a mathematical model based on statistical data series from industry, agriculture, environmental protection, and population in 2000, 2005, 2010, and 2015, this paper aims to estimate the nitrogen income and expenditure of coastal Eastern Guangdong, to reveal the temporal variation of the nitrogen budget in the coastal region with high agriculture intensity, and to suggest a management strategy for the local nitrogen control. The results show that: coastal Eastern Guangdong is a nitrogen surplus region, with nitrogen load and nitrogen flux varying in the range 276.01–299.60 kg N ha−1 yr−1 and 221.26–239.06 kg N ha−1 yr−1, respectively, during the period 2000–2015; from 2000 to 2015, the overall nitrogen surplus and the nitrogen surplus unit area showed an obvious upward trend, indicating that nitrogen pollution in the area was deteriorating; agricultural used fertilizer serves as the main contributor to nitrogen input, while water nitrogen accounts for the highest portion of nitrogen output; despite the fluctuation of nitrogen input and output, water nitrogen output steadily increased, suggesting a stronger water environment management requirement. This research provides reference for researchers and decision-makers in the ecological and environmental domain

Bis(triphenyl­stann­yl) thio­phene-2,5-dicarboxyl­ate

Mol­ecules of the title compound, [Sn2(C6H5)6(C6H2O4S)], lie on inversion centres with the central thio­phene ring disordered equally over two orientations. The carboxyl­ate groups are approximately coplanar with the thio­phene ring [dihedral angle = 4.0 (1)°] and the Sn—O bond distance of 2.058 (4) Å is comparable to that in related organotin carboxyl­ates

2-Amino-4-(4-chloro­phen­yl)-6-ferro­cenylpyridine-3-carbonitrile

In the mol­ecule of the title compound, [Fe(C5H5)(C17H11ClN3)], the dihedral angles between the two five–membered rings and between the two six-membered rings are 3.28 (4) and 51.33 (4)°, respectively. In the crystal structure, inter­molecular N—H⋯N hydrogen bonds link the mol­ecules into centrosymmetric dimers

Chaperone Spy Protects Outer Membrane Proteins from Folding Stress via Dynamic Complex Formation

Gram-negative bacteria have a multicomponent and constitutively active periplasmic chaperone system to ensure the quality control of their outer membrane proteins (OMPs). Recently, OMPs have been identified as a new class of vulnerable targets for antibiotic development, and therefore a comprehensive understanding of OMP quality control network components will be critical for discovering antimicrobials. Here, we demonstrate that the periplasmic chaperone Spy protects certain OMPs against protein-unfolding stress and can functionally compensate for other periplasmic chaperones, namely Skp and FkpA, in the Escherichia coli K-12 MG1655 strain. After extensive; in vivo; genetic experiments for functional characterization of Spy, we use nuclear magnetic resonance and circular dichroism spectroscopy to elucidate the mechanism by which Spy binds and folds two different OMPs. Along with holding OMP substrates in a dynamic conformational ensemble, Spy binding enables OmpX to form a partially folded β-strand secondary structure. The bound OMP experiences temperature-dependent conformational exchange within the chaperone, pointing to a multitude of local dynamics. Our findings thus deepen the understanding of functional compensation among periplasmic chaperones during OMP biogenesis and will promote the development of innovative antimicrobials against pathogenic Gram-negative bacteria.; IMPORTANCE; Outer membrane proteins (OMPs) play critical roles in bacterial pathogenicity and provide a new niche for antibiotic development. A comprehensive understanding of the OMP quality control network will strongly impact antimicrobial discovery. Here, we systematically demonstrate that the periplasmic chaperone Spy has a role in maintaining the homeostasis of certain OMPs. Remarkably, Spy utilizes a unique chaperone mechanism to bind OmpX and allows it to form a partially folded β-strand secondary structure in a dynamic exchange of conformations. This mechanism differs from that of other E. coli periplasmic chaperones such as Skp and SurA, both of which maintain OMPs in disordered conformations. Our study thus deepens the understanding of the complex OMP quality control system and highlights the differences in the mechanisms of ATP-independent chaperones

Effect of Exogenous Nitric Oxide on Postharvest Storage Quality of Hyacinth Bean

In order to study the effect of nitric oxide (NO) on the storage quality of hyacinth bean after harvest, sodium nitroprusside (SNP) was used as an exogenous NO donor in this study. Hyacinth bean was soaked in 0.2 mmol/L SNP solution or distilled water as control for 10 min and then stored at (20 ± 1) ℃ and 80%–90% relative humidity. Decay incidence, rust incidence, hardness, the contents of total soluble solids (TSS), malondialdehyde (MDA), flavonoids, total phenols and chlorophyll, and the activities of antioxidant enzymes (peroxidase (POD), polyphenol oxidase (PPO), phenylalanine ammoniase (PAL), catalase (CAT) and ascorbate peroxidase (APX)) were observed during the storage period. The results showed that exogenous NO treatment could inhibit the rot and rust, keep the color and hardness, and inhibit the degradation of TSS and chlorophyll in hyacinth bean, so that hyacinth bean could maintain good sensory quality. Exogenous NO treatment could also prevent the accumulation of MDA and increase the contents of total phenols and flavonoids. In addition, exogenous NO treatment maintained the activities of PAL, CAT and APX during storage, and inhibited the increase in the activities of POD and PPO, thereby enhancing the antioxidant capacity and delaying the maturation and senescence of hyacinth bean. In conclusion, exogenous NO treatment can delay the postharvest maturation and senescence, maintain the physiological quality during storage, and effectively prolong the shelf life of hyacinth bean