Sulfated and pyruvylated disaccharide alditols obtained from a red seaweed galactan: ESIMS and NMR approaches

The water-soluble acid agaran isolated from Acanthophora spicifera (Rhodophyta) was submitted to alkaline treatment for the complete cyclization of alpha-L-Galp 6-sulfate to 3,6-An-alpha-L-Galp units. The modified agaran was then partially depolymerized using partial reductive hydrolysis. The resulting oligosaccharide mixture was fractionated by adsorption and ion-exchange chromatography. Fractions were purified by gel-filtration chromatography and studied by ESIMS and NMR spectroscopy, including 1D 1H, 13C, DEPT and 2D 1H, 1H COSY, TOCSY and 1H, 13C HMQC procedures. The following neutral, pyruvylated, sulfated and sulfated/pyruvylated disaccharide alditols were obtained: beta-D-Galp-(1-->4)-3,6-An-L-GalOH; 4,6-O-(1-carboxyethylidene)-beta-D-Galp-(1-->4)-3,6-An-L-GalOH; beta-D-Galp 2-sulfate-(1-->4)-3,6-An-L-GalOH and 4,6-O-(1-carboxyethylidene)-beta-D-Galp 2-sulfate-(1-->4)-3,6-An-L-GalOH.     

Role of context in the relationship between form and function: structural plasticity of some PROSITE patterns

True positive hits of PROSITE sequence pattern are expected to have a characteristic three-dimensional structure. The combined sequence-structure attributes of PROSITE patterns can be used for function prediction of an uncharacterized protein with known primary and 3D structure, a situation that might arise in structural genomics projects. We have found specific examples of true hits of PROSITE patterns displaying structural plasticity by assuming significantly different local conformation, depending upon the context. Our work highlights the importance of taking into account all the known distinct conformations of PROSITE patterns, while creating a sensitive 3D template for the pattern, for use in functional annotation.     

Spectropotentiometric analysis of metal binding to structural zinc-binding sites: accounting quantitatively for pH and metal ion buffering effects

Studies of the metal-binding affinity of protein sites are ubiquitous in bioinorganic chemistry and are valuable for the information that they can provide about metal speciation and exchange in biological systems. The potential for error in these studies is high, however, since many competing equilibria are present in solution and must be taken into consideration. Here, we report a new spectropotentiometric titration apparatus that allows pH and UV-vis absorption to be monitored simultaneously on small samples under inert atmosphere. In addition, we explain how data obtained from the complex equilibria can be combined with tabulated information about the protonation and metal-binding constants for common buffers to provide detailed, quantitative information about metal-protein interactions. Application of this approach to the investigation of metal binding to structural zinc-binding domains and common pitfalls encountered when performing these experiments are also discussed. We have used this approach to reevaluate the metal-binding constants of the N-terminal zinc-binding peptide from the HIV-1 nucleocapsid protein (10(-8)M相似文献   

Structural and mechanistic insight from high resolution structures of archaeal rhodopsins

Lipidic cubic phase-grown crystals yielded high resolution structures of a number of archaeal retinal proteins, the molecular mechanisms of which are being revealed as structures of photocycle intermediates become available. The structural basis for bacteriorhodopsin's mechanism of proton pumping is discussed, revealing a well-synchronized sequence of molecular events. Comparison with the high resolution structures of the halide pump halorhodopsin, as well as with the receptor sensory rhodopsin II, illustrates how small and localized structural changes result in functional divergence. Fundamental principles of energy transduction and sensory reception in the archaeal rhodopsins, which may have relevance to other systems, are discussed.     

RIAM and Vinculin Binding to Talin Are Mutually Exclusive and Regulate Adhesion Assembly and Turnover

Talin activates integrins, couples them to F-actin, and recruits vinculin to focal adhesions (FAs). Here, we report the structural characterization of the talin rod: 13 helical bundles (R1–R13) organized into a compact cluster of four-helix bundles (R2–R4) within a linear chain of five-helix bundles. Nine of the bundles contain vinculin-binding sites (VBS); R2R3 are atypical, with each containing two VBS. Talin R2R3 also binds synergistically to RIAM, a Rap1 effector involved in integrin activation. Biochemical and structural data show that vinculin and RIAM binding to R2R3 is mutually exclusive. Moreover, vinculin binding requires domain unfolding, whereas RIAM binds the folded R2R3 double domain. In cells, RIAM is enriched in nascent adhesions at the leading edge whereas vinculin is enriched in FAs. We propose a model in which RIAM binding to R2R3 initially recruits talin to membranes where it activates integrins. As talin engages F-actin, force exerted on R2R3 disrupts RIAM binding and exposes the VBS, which recruit vinculin to stabilize the complex.     

The N-terminal Domain Allosterically Regulates Cleavage and Activation of the Epithelial Sodium Channel

The epithelial sodium channel (ENaC) is activated upon endoproteolytic cleavage of specific segments in the extracellular domains of the α- and γ-subunits. Cleavage is accomplished by intracellular proteases prior to membrane insertion and by surface-expressed or extracellular soluble proteases once ENaC resides at the cell surface. These cleavage events are partially regulated by intracellular signaling through an unknown allosteric mechanism. Here, using a combination of computational and experimental techniques, we show that the intracellular N terminus of γ-ENaC undergoes secondary structural transitions upon interaction with phosphoinositides. From ab initio folding simulations of the N termini in the presence and absence of phosphatidylinositol 4,5-bisphosphate (PIP₂), we found that PIP₂ increases α-helical propensity in the N terminus of γ-ENaC. Electrophysiology and mutation experiments revealed that a highly conserved cluster of lysines in the γ-ENaC N terminus regulates accessibility of extracellular cleavage sites in γ-ENaC. We also show that conditions that decrease PIP₂ or enhance ubiquitination sharply limit access of the γ-ENaC extracellular domain to proteases. Further, the efficiency of allosteric control of ENaC proteolysis is dependent on Tyr³⁷⁰ in γ-ENaC. Our findings provide an allosteric mechanism for ENaC activation regulated by the N termini and sheds light on a potential general mechanism of channel and receptor activation.     

Turning natural products into insecticide candidates: Design and semisynthesis of novel fraxinellone-based N-(1,3-thiazol-2-yl)carboxamides against two crop-threatening insect pests

To improve the insecticidal activities of fraxinellone, two series of fraxinellone-based N-(1,3-thiazol-2-yl)carboxamides containing 25 compounds were prepared by structural modification. Their structures were determined by melting point, optical rotation, IR, ¹H NMR and ESI-MS. The steric configurations of compounds 6i, 7d and 7i were unambiguously confirmed by X-ray diffraction further. The bioassay showed that compounds 6b and 6i exhibited more potent larvicidal and growth inhibitory activities against Plutella xylostella Linnaeus and Mythimna separata Walker, respectively. Moreover, compounds 6b and 6i also displayed low cytotoxicity to noncancerous mammalian cells. The structure–activity relationships (SARs) of all target compounds were also observed.     

Revisiting microtubule targeting agents: α-Tubulin and the pironetin binding site as unexplored targets for cancer therapeutics

Molecules that bind to tubulin and disrupt tubulin dynamics are known as microtubule targeting agents. Treatment with a microtubule targeting agent leads to cell cycle arrest followed by apoptosis. Tubulin inhibitors have been highly effective in the clinical treatment of a variety of tumors and are being investigated for treatment of several other diseases. Currently, all FDA approved microtubule inhibitors bind to β-tubulin. Given the overall success of tubulin-binding agents in anticancer chemotherapy, α-tubulin is an attractive and unexplored target. Herein, we will discuss pironetin, the only compound known to bind α-tubulin, with particular focus on the known biological properties, the total syntheses, exploration of its structure-activity relationship, and future directions.     

Read count-based method for high-throughput allelic genotyping of transposable elements and structural variants

Background

Like other structural variants, transposable element insertions can be highly polymorphic across individuals. Their functional impact, however, remains poorly understood. Current genome-wide approaches for genotyping insertion-site polymorphisms based on targeted or whole-genome sequencing remain very expensive and can lack accuracy, hence new large-scale genotyping methods are needed.

Results

We describe a high-throughput method for genotyping transposable element insertions and other types of structural variants that can be assayed by breakpoint PCR. The method relies on next-generation sequencing of multiplex, site-specific PCR amplification products and read count-based genotype calls. We show that this method is flexible, efficient (it does not require rounds of optimization), cost-effective and highly accurate.

Conclusions

This method can benefit a wide range of applications from the routine genotyping of animal and plant populations to the functional study of structural variants in humans.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1700-4) contains supplementary material, which is available to authorized users.     

Photo-induced inhibition of insulin amyloid fibrillation on online laser measurement

Protein aggregation and amyloid fibrillation can lead to several serious diseases and protein drugs ineffectiveness; thus, the detection and inhibition of these processes have been of great interest. In the present study, the inhibition of insulin amyloid fibrillation by laser irradiation was investigated using dynamic light scattering (DLS), transmission electron microscopy (TEM), far-UV circular dichroism (far-UV CD), and thioflavin T (ThT) fluorescence. During heat-induced aggregation, the size distribution of two insulin solutions obtained by online and offline dynamic light scattering were different. The laser-on insulin in the presence of 0.1 M NaCl exhibited fewer fibrils than the laser-off insulin, whereas no insulin fibril under laser irradiation was observed in the absence of 0.1 M NaCl for 45 h incubation. Moreover, our CD results showed that the laser-irradiated insulin solution maintained mainly an α-helical conformation, but the laser-off insulin solution formed bulk fibrils followed by a significant increase in β-sheet content for 106 h incubation. These findings provide an inhibition method for insulin amyloid fibrillation using the laser irradiation and demonstrate that the online long-time laser measurements should be carefully used in the study of amyloid proteins because they may change the original results.