Oligomeric structure of the ATP-dependent protease La (Lon) of Escherichia coli

Lon, also known as protease La, belongs to a class of ATP-dependent serine protease. It plays an essential role in degradation of abnormal proteins and of certain short-lived regulatory proteins, and is thought to possess a Ser-Lys catalytic dyad. To examine the structural organization of Lon, we performed an electron microscope analysis. The averaged images of Lon with end-on orientation revealed a six-membered, ring-shaped structure with a central cavity. The side-on view showed a two-layered structure with an equal distribution of mass across the equatorial plane of the complex. Since a Lon subunit possesses two large regions containing nucleotide binding and proteolytic domains, each layer of the Lon hexamer appears to consist of the side projections of one of the major domains arranged in a ring. Lon showed a strong tendency to form hexamers in the presence of Mg(2+), but dissociated into monomers and/or dimers in its absence. Moreover, Mg(2+)-dependent hexamer formation was independent of ATP. These results indicate that Lon has a hexameric ring-shaped structure with a central cavity, and that the establishment of this configuration requires Mg(2+), but not ATP.     

Alternative complex formation of the Ca-regulated protein kinase CIPK1 controls abscisic acid-dependent and independent stress responses in Arabidopsis

Intracellular release of calcium ions belongs to the earliest events in cellular stress perception. The molecular mechanisms integrating signals from different environmental cues and translating them into an optimized response are largely unknown. We report here the functional characterization of CIPK1, a protein kinase interacting strongly with the calcium sensors CBL1 and CBL9. Comparison of the expression patterns indicates that the three proteins execute their functions in the same tissues. Physical interaction of CIPK1 with CBL1 and CBL9 targets the kinase to the plasma membrane. We show that, similarly to loss of CBL9 function, mutation of either CBL1 or CIPK1 renders plants hypersensitive to osmotic stress. Remarkably, in contrast to the cbl1 mutant and similarly to the cbl9 mutant, loss of CIPK1 function impairs abscisic acid (ABA) responsiveness. We therefore suggest that, by alternative complex formation with either CBL1 or CBL9, the kinase CIPK1 represents a convergence point for ABA-dependent and ABA-independent stress responses. Based on our genetic, physiological and protein-protein interaction data, we propose a general model for information processing in calcium-regulated signalling networks.     

Feasibility of hydrogen production in thermophilic mixed fermentation by natural anaerobes

The biological sludge from an animal wastewater treatment plant was treated to enrich hydrogen-producing mixed bacteria, and effects on hydrogen yield were investigated during anaerobic fermentation at 55 degrees C. Enrichment of hydrogen-producing bacteria was conducted at pH adjustment of inocula to 3 and 5 with and without additional heat treatment (NHT and HT). The enriched mixed bacteria were cultivated at initial pHs of 5, 6, and 7 with synthetic organic wastewater containing different levels of nitrogen (2.0 and 0.8 g/l as total nitrogen) under static batch conditions. The main effects of heat treatment and enrichment pH were significant on hydrogen production. There was no significant effect of different nitrogen concentrations on hydrogen production. The methane-free biogas contained hydrogen levels of up to 64% for a fermentative condition that showed maximum hydrogen evolution (at culture pH 5 after enrichment at pH 5 with HT). The dominating intermediate metabolites were acetate, n-butyrate, and ethanol. Yields of produced hydrogen were significantly dependent upon levels of n-butyrate.     

Crystallization,molecular replacement solution,and refinement of tetrameric β-amylase from sweet potato

Sweet potato β-amylase is a tetramer of identical subunits, which are arranged to exhibit 222 molecular symmetry. Its subunit consists of 498 amino acid residues (Mr 55,880). It has been crystallized at room temperature using polyethylene glycol 1500 as precipitant. The crystals, growing to dimensions of 0.4 mm × 0.4 mm × 1.0 mm within 2 weeks, belong to the tetragonal space group P4₂2₁2 with unit cell dimensions of a = b = 129.63 Å and c = 68.42 Å. The asymmetric unit contains 1 subunit of β-amylase, with a crystal volume per protein mass (V_M) of 2.57 ų/Da and a solvent content of 52% by volume. The three-dimensional structure of the tetrameric β-amylase from sweet potato has been determined by molecular replacement methods using the monomeric structure of soybean enzyme as the starting model. The refined subunit model contains 3,863 nonhydrogen protein atoms (488 amino acid residues) and 319 water oxygen atoms. The current R-value is 20.3% for data in the resolution range of 8–2.3 Å (with 2 σ cut-off) with good stereochemistry. The subunit structure of sweet potato β-amylase (crystallized in the absence of α-cyclodextrin) is very similar to that of soybean β-amylase (complexed with α-cyclodextrin). The root-mean-square (RMS) difference for 487 equivalent Cα atoms of the two β-amylases is 0.96 Å. Each subunit of sweet potato β-amylase is composed of a large (α/β)₈ core domain, a small one made up of three long loops [L3 (residues 91–150), LA (residues 183–258), and L5 (residues 300–327)], and a long C-terminal loop formed by residues 445–493. Conserved Glu 187, believed to play an important role in catalysis, is located at the cleft between the (α/β)₈ barrel core and a small domain made up of three long loops (L3, L4, and L5). Conserved Cys 96, important in the inactivation of enzyme activity by sulfhydryl reagents, is located at the entrance of the (α/β)₈ barrel. © 1995 Wiley-Liss, Inc.     

Characterization of binding proteins that recognize oligoglucoside elicitors of phytoalexin synthesis in soybean

We are studying the cellular signaling pathway leading to pterocarpan phytoalexin biosynthesis in soybean that is induced by a branched hepta-β-glucoside originally isolated from the mycelial walls of the phytopathogenic oomycete Phytophthora sojae. Our research has focused on the specific recognition of the hepta-β-glucoside elicitor by binding proteins in soybean cells. Elicitor-binding proteins with properties expected of physiological receptors for the hepta-β-glucoside elicitor have been identified in soybean root membranes. These elicitor-binding proteins co-migrate with a plasma membrane marker (vanadate-sensitive H⁺-ATPase) on linear sucrose density gradients. Binding of a radio-iodinated derivative of the hepta-β-glucoside elicitor by membrane-localized elicitor-binding proteins is specific, reversible, saturable, and of high affinity (K_d? 1 nM). After solubilization with the nonionic detergent, n-dodecylsucrose, the elicitor-binding proteins retain their high affinity (K_d= 1.8 nM) for the radiolabeled elicitor and their binding specificity for elicitor-active oligoglucosides. A direct correlation is observed between the ability of oligoglucosides to displace labeled elicitor from the elicitor-binding proteins and the elicitor activity of the oligosaccharides. Thus, the elicitor-binding proteins recognize the same structural elements of the hepta-β-glucoside elicitor that are essential for its phytoalexin-inducing activity, suggesting that the binding proteins are physiological receptors for the elicitor. Current research is directed toward the purification of the hepta-β-glucoside elicitor-binding proteins by using ligand affinity chromatography. Purification and characterization of the hepta-β-glucoside binding proteins are among the first steps toward elucidating how the hepta-β-glucoside elicitor triggers the signal transduction pathway that ultimately leads to the synthesis of phytoalexins in soybean.     

Universal Strategy with Structural and Chemical Crosslinking Interface for Efficient and Stable Perovskite Solar Cells

Due to the limited interface contact and weak interfacial interaction, planar heterojunction perovskite solar cells (PSCs) have space for further improvement. Herein, a structural and chemical crosslinking interface is proposed and constructed by introducing an extra layer, which blends tin dioxide (SnO₂) nanoparticles with chloride salts. Since the incorporated materials can be dissolved during the fabrication of perovskite, the quality of perovskite films is improved, leading to larger grain size and reduced trap-state density. Also, more chloride ions at the SnO₂/perovskite interface are observed and the interaction between Cl⁻ and Sn⁴⁺ is confirmed. It results in more pronounced n-type SnO₂ with better conductivity and deeper conduction bands, leading to preferable energy level alignment between SnO₂ and perovskite. Consequently, the open-circuit voltage and fill factor of the devices increase, and target cells present better stability, retaining 98% of initial efficiencies after >10 000 h storage in dry air (≈5% relative humidity) and maintaining 85.50% of the initial efficiency after 1000 h of operation under light. This strategy enables the achievement of 25.28% efficiency with a low bandgap (1.53 eV) perovskite composition, and it is confirmed to be universal when other related materials are utilized.     

Expression and characterization of the integral membrane domain of bacterial histidine kinase SCO3062 for structural studies

Bacterial histidine kinases play an important role in the response to external stimuli. Structural studies of the histidine kinase transmembrane domain are challenging due to difficulties in protein expression and sample preparation. After carrying out expression screening of a series of histidine kinases, we investigated sample preparation methods for obtaining high quality samples of the periplasmic and transmembrane domain (PTD) of the bacterial histidine kinase SCO3062. Various sample conditions were tested for their ability to give homogeneous NMR spectra of the SCO3062 PTD with well-resolved resonances. Circular dichroism and 3D ¹⁵N-edited NOESY spectrum results demonstrate that the SCO3062 PTD is predominantly α-helical. This method should be applicable to the NMR analysis of other transmembrane proteins.     

Association of CHRDL1 Mutations and Variants with X-linked Megalocornea,Neuh?user Syndrome and Central Corneal Thickness

We describe novel CHRDL1 mutations in ten families with X-linked megalocornea (MGC1). Our mutation-positive cohort enabled us to establish ultrasonography as a reliable clinical diagnostic tool to distinguish between MGC1 and primary congenital glaucoma (PCG). Megalocornea is also a feature of Neuhäuser or megalocornea-mental retardation (MMR) syndrome, a rare condition of unknown etiology. In a male patient diagnosed with MMR, we performed targeted and whole exome sequencing (WES) and identified a novel missense mutation in CHRDL1 that accounts for his MGC1 phenotype but not his non-ocular features. This finding suggests that MMR syndrome, in some cases, may be di- or multigenic. MGC1 patients have reduced central corneal thickness (CCT); however no X-linked loci have been associated with CCT, possibly because the majority of genome-wide association studies (GWAS) overlook the X-chromosome. We therefore explored whether variants on the X-chromosome are associated with CCT. We found rs149956316, in intron 6 of CHRDL1, to be the most significantly associated single nucleotide polymorphism (SNP) (p = 6.81×10⁻⁶) on the X-chromosome. However, this association was not replicated in a smaller subset of whole genome sequenced samples. This study highlights the importance of including X-chromosome SNP data in GWAS to identify potential loci associated with quantitative traits or disease risk.     

Evaluation of Adult White Sturgeon Swimming Capabilities and Applications to Fishway Design

Concern over passage of sturgeon barriers, has focused attention on fishway design that accommodates its swimming performance. In order to evaluate swimming performance, regarding fish ladder type partial barriers, wild adult sturgeons, Acipenser transmontanus; 121–76m fork length, were captured in the San Francisco Bay Estuary and Yolo Bypass toe drain. Hydrodynamic forces and kinematic parameters for swimming performance data were collected in a laboratory flume under three flow conditions through barriers and ramp. The experiments were conducted in a 24.4 m long, 2.1 m wide, and 1.62 m deep aluminum channel. Two geometric configurations of the laboratory model were designed based on channel characteristics that have been identified in natural river systems. At a given swimming speed and fish size, the highest guidance efficiencies of successful white sturgeon passage as a function of flow depth, flow velocity, turbulence intensity, Reynolds number, Froude number and shear velocity observed in the steady flow condition, tested with the horizontal ramp structure, occurred at an approach velocity of 0.33 ms^-1. The guidance efficiency of successful sturgeon passage increased both with increasing flow velocity and Froude number, and decreased both with the flow depth and the turbulence intensity. This study also provides evidence that tail beat frequency increases significantly with swimming speed, but tail beat frequency decreases with fish total length. Stride length increases both with swimming speed and fish total length. The importance of unsteady forces is expressed by the reduced frequency both with swimming speed and fish total length. Regression analysis indicates that swimming kinematic variables are explained by the swimming speed, the reduced frequency and the fish total length. The results emphasize the importance of fish ladder type patchiness when a fishway is designed for the passage of sturgeon.