Structure–function relationships of four compression wood types: micromechanical properties at the tissue and fibre level

The mechanisms behind compressive stress generation in gymnosperms are not yet fully understood. Investigating the structure–function relationships at the tissue and cell level, however, can provide new insights. Severe compression wood of all species lacks a S3 layer, has a high microfibril angle in the S2 layer and a high lignin content. Additionally, special features like helical cavities or spiral thickenings appear, which are not well understood in terms of their mechanical relevance, but need to be examined with regard to evolutionary trends in compression wood development. Thin compression wood foils and isolated tracheids of four gymnosperm species [Ginkgo biloba L., Taxus baccata L., Juniperus virginiana L., Picea abies (L.) Karst.] were investigated. The tracheids were isolated mechanically by peeling them out of the solid wood using fine tweezers. In contrast to chemical macerations, the cell wall components remained in their original condition. Tensile properties of tissue foils and tracheids were measured in a microtensile apparatus under wet conditions. Our results clearly show an evolutionary trend to a much more flexible compression wood. An interpretation with respect to compressive stress generation is discussed.     

The DEK protein--an abundant and ubiquitous constituent of mammalian chromatin

The protein DEK is an abundant and ubiquitous chromatin protein in multicellular organisms (not in yeast). It is expressed in more than a million copies/nucleus of rapidly proliferating mammalian cells. DEK has two DNA binding modules of which one includes a SAP box, a sequence motif that DEK shares with a number of other chromatin proteins. DEK has no apparent affinity to specific DNA sequences, but preferentially binds to superhelical and cruciform DNA, and induces positive supercoils into closed circular DNA. The available evidence strongly suggests that DEK could function as an architectural protein in chromatin comparable to the better known classic architectural chromatin proteins, the high-mobility group or HMG proteins.     

Adaptive Growth of Gymnosperm Branches-Ultrastructural and Micromechanical Examinations

The top, the lateral and the underside of basal branch segments of two gymnosperm species, spruce (Picea abies [L.] Karst.) and yew (Taxus baccata L.), were studied with respect to possible adaptation in structural and mechanical properties. Microtensile tests were performed on thin wet foils, which were removed from the periphery of the branches. Structural parameters such as density and the microfibril angle in the S2-layer were examined to investigate the structure-function relationships of the branch wood. The top, the lateral and the underside of both branches showed significant differences in their structural and mechanical properties. However, no significant variations were observed as a function of age and size development. The findings were discussed in view of adaptive growth strategies of trees, including biomechanical constraints of a horizontally growing branch.     

Removal of Ca2+ channel beta3 subunit enhances Ca2+ oscillation frequency and insulin exocytosis

An oscillatory increase in pancreatic beta cell cytoplasmic free Ca2+ concentration, [Ca2+]i, is a key feature in glucose-induced insulin release. The role of the voltage-gated Ca2+ channel beta3 subunit in the molecular regulation of these [Ca2+]i oscillations has now been clarified by using beta3 subunit-deficient beta cells. beta3 knockout mice showed a more efficient glucose homeostasis compared to wild-type mice due to increased glucose-stimulated insulin secretion. This resulted from an increased glucose-induced [Ca2+]i oscillation frequency in beta cells lacking the beta3 subunit, an effect accounted for by enhanced formation of inositol 1,4,5-trisphosphate (InsP3) and increased Ca2+ mobilization from intracellular stores. Hence, the beta3 subunit negatively modulated InsP3-induced Ca2+ release, which is not paralleled by any effect on the voltage-gated L type Ca2+ channel. Since the increase in insulin release was manifested only at high glucose concentrations, blocking the beta3 subunit in the beta cell may constitute the basis for a novel diabetes therapy.     

Structural basis of dynamic glycine receptor clustering by gephyrin

Gephyrin is a bi-functional modular protein involved in molybdenum cofactor biosynthesis and in postsynaptic clustering of inhibitory glycine receptors (GlyRs). Here, we show that full-length gephyrin is a trimer and that its proteolysis in vitro causes the spontaneous dimerization of its C-terminal region (gephyrin-E), which binds a GlyR beta-subunit-derived peptide with high and low affinity. The crystal structure of the tetra-domain gephyrin-E in complex with the beta-peptide bound to domain IV indicates how membrane-embedded GlyRs may interact with subsynaptic gephyrin. In vitro, trimeric full-length gephyrin forms a network upon lowering the pH, and this process can be reversed to produce stable full-length dimeric gephyrin. Our data suggest a mechanism by which induced conformational transitions of trimeric gephyrin may generate a reversible postsynaptic scaffold for GlyR recruitment, which allows for dynamic receptor movement in and out of postsynaptic GlyR clusters, and thus for synaptic plasticity.     

Structure of the periplasmic chaperone Skp suggests functional similarity with cytosolic chaperones despite differing architecture

The 17-kDa protein (Skp) of Escherichia coli is a homotrimeric periplasmic chaperone for newly synthesized outer-membrane proteins. Here we present its X-ray structure at a resolution of 2.35 A. Three hairpin-shaped alpha-helical extensions reach out by approximately 60 A from a trimerization domain, which is composed of three intersubunit beta-sheets that wind around a central axis. The alpha-helical extensions approach each other at their distal turns, resulting in a fold that resembles a 'three-pronged grasping forceps'. The overall shape of Skp is reminiscent of the cytosolic chaperone prefoldin, although it is based on a radically different topology. The peculiar architecture, with apparent plasticity of the prongs and distinct electrostatic and hydrophobic surface properties, supports the recently proposed biochemical mechanism of this chaperone: formation of a Skp(3)-Omp complex protects the outer membrane protein from aggregation during passage through the bacterial periplasm.     

Identification of the Anabaena sp. strain PCC7120 cyanophycin synthetase as suitable enzyme for production of cyanophycin in gram-negative bacteria like Pseudomonas putida and Ralstonia eutropha

The cyanophycin synthetase gene cphA1 encoding the major cyanophycin synthetase (CphA) of Anabaena sp. strain PCC7120 was expressed in Escherichia coli conferring so far the highest specific CphA activity to E. coli (6.7 nmol arginine per min and mg protein). CphA1 and cphA genes of Synechocystis sp. strains PCC6803 and PCC6308 and Synechococcus strain MA19 were also expressed in wild types and polyhydroxyalkanoate-negative (PHA) mutants of Pseudomonas putida and Ralstonia eutropha. Recombinant strains of these bacteria expressing cphA1 accumulated generally more cyanophycin (23.0 and 20.0% of cellular dry matter, CDM, respectively) than recombinants expressing any other cphA (6.8, 9.0, or 15.8% of CDM for P. putida strains and 7.3, 12.6, or 14.1% of CDM for R. eutropha). Furthermore, PHA-negative mutants of P. putida (9.7, 10.0, 17.5, or 24.0% of CDM) and R. eutropha (8.9, 13.8, 16.0, or 22.0% of CDM) accumulated generally more cyanophycin than the corresponding PHA-positive parent strains (6.8, 9.0, 15.8, and 23.0% of CDM for P. putida strains and 7.3, 12.6, 14.1, or 20.0% of CDM for R. eutropha strains). Recombinant strains of Gram-positive bacteria (Bacillus megaterium, Corynebacterium glutamicum) were not suitable for cyanophycin production due to accumulation of less cyanophycin and retarded release of cyanophycin. PHA-negative mutants of P. putida and R. eutropha expressing cphA1 of Anabaena sp. strain PCC7120 are therefore preferred candidates for industrial production of cyanophycin.     

A molecular docking study of estrogenically active compounds with 1,2-diarylethane and 1,2-diarylethene pharmacophores

Numerous selective estrogen receptor modulators (SERMs) have been synthesized and assayed in recent years. The focus of this study is to apply coarse-grain molecular docking procedures coupled with fine-grain all-atom force field optimization strategies to shed light on the binding mechanisms of currently available estrogen receptor-active compounds. Although the mechanics of ligand binding in estrogen receptors is generally well understood, there is room for surprises. In this paper computational evidence corroborating the experimentally observed type I agonistic binding mode for estradiol (E2) and diethylstilbesterol (DES) and the type II antagonistic binding mode for 4-hydroxytamoxifen and raloxifen is presented. Included in this type I agonistic mode are the DES derivatives, transstilbene and 1,2-diaryldiaminoethane. In addition, a novel ‘type II agonistic’ binding mode for 2,3-diarylimidazolines, 4,5-diarylimidazoles, 2,3-diarylpiperazines is introduced. This mode is stabilized by suggesting alternative hydrogen bond anchor points in the ligand binding domain as potential leads for future drug design.     

Structural mechanism of specific ligand recognition by a lipocalin tailored for the complexation of digoxigenin

DigA16 is an artificial digoxigenin-binding protein, which was derived from the bilin-binding protein, a lipocalin of Pieris brassicae, via reshaping of its natural ligand pocket. Here we report the crystal structures of DigA16 in the presence of either digoxigenin or digitoxigenin and for the apo-protein at resolutions below 1.9A. As a consequence of the altogether 17 amino acid substitutions within the binding site significant structural changes have occurred in the four loops that form the entrance to the ligand pocket on top of the structurally conserved beta-barrel framework. For example, one loop adopts a new alpha-helical backbone structure, which seems to be induced by few critical side-chain contacts. Digoxigenin becomes almost fully buried (by 95%) upon complexation, whereby specificity for the hydrophilic steroid is maintained through hydrogen-bonding networks and shape complementarity. The differential binding of the related steroid digitoxigenin is mainly governed by an internal histidine residue, whose side-chain undergoes significant induced fit. Among those amino acids that line the ligand pocket two tyrosine and one tryptophan residue provide the largest contacts. Interestingly, corresponding three side-chains are found with the same mutual orientation in the anti-digoxigenin antibody 26-10, even though the hapten orientation is quite different there and only 66% of the steroid surface is buried in the combining site. Hence, in the case of the engineered lipocalin DigA16 an example of convergent in vitro evolution is observed. Generally, the remarkable structural plasticity of the loop region and the role of polar residues in the binding site illustrate the potential of the lipocalin scaffold for the generation of specific receptor proteins towards a variety of ligands.