Investigating the permanent electric dipole moment of beta-lactoglobulin fibrils, using transient electric birefringence

Amyloid fibrils, which are polymeric assemblies of protein molecules, have been intensively studied on a structural level, yet due to complications such as the disorder within the molecules, several aspects of their structure remain mysterious. Similarly, the kinetics of assembly are not well understood. Here we investigate the electric dipole moment of beta-lactoglobulin fibrils, a model amyloid fibril system, by applying the technique of transient electric birefringence. This moment appears to be large, and comparable to the total moment of the constituent protein monomers if they were joined in a chain, head-to-tail, without changing conformation, suggesting an ordered joining of monomers in the fibril. Such an ordered assembly may have implications for the assembly mechanism of beta-lactoglobulin fibrils in particular, and amyloid fibrils in general.     

Studies toward a conjugate vaccine for anthrax. Synthesis and characterization of anthrose [4,6-dideoxy-4-(3-hydroxy-3-methylbutanamido)-2-O-methyl-D-glucopyranose] and its methyl glycosides

The key step in the first chemical synthesis of anthrose (16) and its methyl alpha- (6) and beta-glycoside (22) was inversion of configuration at C-2 in triflates 10, 2, and 18, respectively, obtained from the common intermediate, methyl 4-azido-3-O-benzyl-4,6-dideoxy-alpha-D-mannopyranoside (1). To prepare methyl alpha-anthroside (6), methylation at O-2 of the gluco product 3, obtained from 2, was followed by hydrogenation/hydrogenolysis of the formed 2-methyl ether 4, to simultaneously remove the protecting benzyl group and reduce the azido function. Subsequent N-acylation of the formed amine 5 with 3-hydroxy-3-methylbutyric acid gave the target methyl alpha-glycoside 6. Synthesis of methyl beta-anthroside (22) comprised the same sequence of reactions, starting from the known methyl 4-azido-3-O-benzyl-4,6-dideoxy-beta-D-mannopyranoside (17), which was prepared from 1. In the synthesis of anthrose (16), 1-thio-beta-glucoside 11, obtained from 1 through 10, was methylated at O-2, and the azido function in the resulting benzylated 1-thioglycoside 12 was selectively reduced to give amine 13. After N-acylation with 3-hydroxy-3-methylbutyric acid, 1-thioglycoside 14 was hydrolyzed to give the corresponding reducing sugar, aldol 15, which was debenzylated to afford anthrose.     

Kinin-mediated inflammation in neurodegenerative disorders

The mediatory role of kinins in both acute and chronic inflammation within nervous tissues has been widely described. Bradykinin, the major representative of these bioactive peptides, is one of a few mediators of inflammation that directly stimulates afferent nerves due to the broad expression of specific kinin receptors in cell types in these tissues. Moreover, kinins may be delivered to a site of injury not only after their production at the endothelium surface but also following their local production through the enzymatic degradation of kininogens at the surface of nerve cells. A strong correlation between inflammatory processes and neurodegeneration has been established. The activation of nerve cells, particularly microglia, in response to injury, trauma or infection initiates a number of reactions in the neuronal neighborhood that can lead to cell death after the prolonged action of inflammatory substances. In recent years, there has been a growing interest in the effects of kinins on neuronal destruction. In these studies, the overexpression of proteins involved in kinin generation or of kinin receptors has been observed in several neurologic disorders including neurodegenerative diseases such Alzheimer's disease and multiple sclerosis as well as disorders associated with a deficiency in cell communication such as epilepsy. This review is focused on recent findings that provide reliable evidence of the mediatory role of kinins in the inflammatory responses associated with different neurological disorders. A deeper understanding of the role of kinins in neurodegenerative diseases is likely to promote the future development of new therapeutic strategies for the control of these disorders. An example of this could be the prospective use of kinin receptor antagonists.     

Three-dimensional structure of the inclusion complex between phloridzin and beta-cyclodextrin

The inclusion of phloridzin into beta-cyclodextrin was studied as a model of molecular recognition in membranes. Effects on 1H NMR spectra and NOE correlational peaks between phloridzin and beta-cyclodextrin were observed in the complex. Strong NOEs were observed between hydrogens of a phenol group in phloridzin and beta-cyclodextrin. The three-dimensional structure of the inclusion complex between phloridzin and beta-cyclodextrin was simulated with distance constraints estimated by the intensity of NOE peaks using the DADAS90 programs. Two inclusion possibilities were suggested-the large rim of beta-cyclodextrin as an entrance of the inclusion and the small rim of beta-cyclodextrin as the entrance. In both cases, the phenol group of phloridzin was included in the hydrophobic space of beta-cyclodextrin.     

N-Terminal pyroglutamate formation of Aβ38 and Aβ40 enforces oligomer formation and potency to disrupt hippocampal long-term potentiation

Pyroglutamate (pGlu)-modified amyloid peptides have been identified in sporadic and familial forms of Alzheimer's disease (AD) and the inherited disorders familial British and Danish Dementia (FBD and FDD). In this study, we characterized the aggregation of amyloid-β protein Aβ37, Aβ38, Aβ40, Aβ42 and ADan species in vitro, which were modified by N-terminal pGlu (pGlu-Aβ3-x, pGlu-ADan) or possess the intact N-terminus (Aβ1-x, ADan). The pGlu-modification confers rapid formation of oligomers and short fibrillar aggregates. In accordance with these observations, the pGlu-modified Aβ38, Αβ40 and Αβ42 species inhibit hippocampal long term potentiation of synaptic response, but pGlu-Aβ3-42 showing the highest effect. Among the unmodified Aβ peptides, only Aβ1-42 exhibites such propensity, which was similar to pGlu-Aβ3-38 and pGlu-Aβ3-40. Likewise, the amyloidogenic peptide pGlu-ADan impaired synaptic potentiation more pronounced than N-terminal unmodified ADan. The results were validated using conditioned media from cultivated HEK293 cells, which express APP variants favoring the formation of Aβ1-x, Aβ3-x or N-truncated pGlu-Aβ3-x species. Hence, we show that the ability of different amyloid peptides to impair synaptic function apparently correlates to their potential to form oligomers as a common mechanism. The pGlu-modification is apparently mediating a higher surface hydrophobicity, as shown by 1-anilinonaphtalene-8-sulfonate fluorescence, which enforces potential to interfere with neuronal physiology.     

Psb30 contributes to structurally stabilise the Photosystem II complex in the thermophilic cyanobacterium Thermosynechococcus elongatus

A deletion mutant that lacks the Psb30 protein, one of the small subunits of Photosystem II, was constructed in a Thermosynechococcus elongatus strain in which the D1 protein is expressed from the psbA₃ gene (WT*). The ΔPsb30 mutant appears more susceptible to photodamage, has a cytochrome b₅₅₉ that is converted into the low potential form, and probably also lacks the PsbY subunit. In the presence of an inhibitor of protein synthesis, the ?Psb30 lost more rapidly the water oxidation function than the WT* under the high light conditions. These results suggest that Psb30 contributes to structurally and functionally stabilise the Photosystem II complex in preventing the conversion of cytochrome b₅₅₉ into the low potential form. Structural reasons for such effects are discussed.     

Phenylalanine promotes interaction of transmembrane domains via GxxxG motifs

Interactions of transmembrane helices play a crucial role in the folding and oligomerisation of integral membrane proteins. In order to uncover novel sequence motifs mediating these interactions, we randomised one face of a transmembrane helix with a set of non-polar or moderately polar amino acids. Those sequences capable of self-interaction upon integration into bacterial inner membranes were selected by means of the ToxR/POSSYCCAT system. A comparison between low/medium-affinity and high-affinity sequences reveals that high-affinity sequences are strongly enriched in phenylalanine residues that are frequently observed at the − 3 position of GxxxG motifs, thus yielding FxxGxxxG motifs. Mutation of Phe or GxxxG in selected sequences significantly reduces self-interaction of the transmembrane domains without affecting their efficiency of membrane integration. Conversely, grafting FxxGxxxG onto unrelated transmembrane domains strongly enhances their interaction. Further, we find that FxxGxxxG is significantly over-represented in transmembrane domains of bitopic membrane proteins. The same motif contributes to self-interaction of the vesicular stomatitis virus G protein transmembrane domain. We conclude that Phe stabilises membrane-spanning GxxxG motifs. This is one example of how the role of certain side-chains in helix-helix interfaces is modulated by sequence context.     

Latex extractables of Calotropis gigantea

The hexane and methanol soluble extract of the latex coagulum of Calotropis gigantea afforded two new triterpene esters, viz. 3′-methylbutanoates of α-amyrin and ψ-taraxasterol, besides the known 3′-methylbutanoates of three triterpene alcohols. The compositions of the alkane fraction, total triterpene alcohol fraction, and free, acetyl and 3′-methylbutanoyl triterpene alcohol fractions of the extract were determined.     

Substrate-induced double sided H-bond network as a means of domain closure in 3-phosphoglycerate kinase

Closure of the two domains of 3-phosphoglycerate kinase, upon substrate binding, is essential for the enzyme function. The available crystal structures cannot provide sufficient information about the mechanism of substrate assisted domain closure and about the requirement of only one or both substrates, since lattice forces may hinder the large scale domain movements. In this study the known X-ray data, obtained for the open and closed conformations, were probed by solution small-angle X-ray scattering experiments. The results prove that binding of both substrates is essential for domain closure. Molecular graphical analysis, indeed, reveals formation of a double-sided H-bond network, which affects substantially the shape of the main molecular hinge at beta-strand L, under the concerted action of both substrates.