Beta-sheet Content Research Articles

Cellular distribution of activity for three enzymes with maltose binding protein as fusion partner and the structural implications

The bacterial SEC pathway is commonly used for secretion of heterologous proteins in E. coli by fusing them to transported proteins to facilitate downstream processing. While some proteins are translocated very efficiently, some reside in the cytoplasm. In this work, maltose binding protein (MBP) was fused to 3 cytoplamic enzymes from Thermus thermophilus (serine protease, 251 residues; glucose isomerase, 381 residues; pullulanase, 718 residues) to study the protein transport from the cytoplasm by quantifying the distribution of activities in different cellular compartments. Pullulanase activity was harvested exclusively in the periplasm; however, glucose isomerase activity was harvested exclusively in the cytoplasm. Considerable serine protease activity was found in the periplasm, but after 10 h of induction activity dropped sharply and no activity was found thereafter in either compartment. This was attributed to the instability of the plasmid probably caused by the proteolytic activity of the protease Computations of hypothetical folding rates and secondary structure contents of the proteins showed that folding rates, in addition to alpha-helix and beta-sheet contents of proteins, could be important determinants for efficient translocation by the SEC pathway. These results may give clues to predict whether a protein would be a suitable fusion tail for periplasmic transport with MBP.

Influence of bovine serum albumin on the secondary structure of interferon alpha 2b as determined by far UV circular dichroism spectropolarimetry

Many therapeutic biologics are formulated with excipients, including the protein excipient human serum albumin (HSA), to increase stability and prevent protein aggregation and adsorption onto glass vials. One biologic formulated with albumin is interferon alpha-2b (IFN alpha-2b). As is the case with other therapeutic biologics, the increased structural complexity of IFN alpha-2b compared to a small molecule drug requires that both the correct chemical structure (amino acid sequence) and also the correct secondary and tertiary structures (3 dimensional fold) be verified to assure safety and efficacy. Although numerous techniques are available to assess a biologic's primary, secondary and tertiary structures, difficulties arise when assessing higher order structure in the presence of protein excipients. In these studies far UV circular dichroism spectropolarimetry (far UV-CD) was used to determine the secondary structure of IFN alpha-2b in the presence of a protein excipient (bovine serum albumin, BSA). We demonstrated that the secondary structure of IFN alpha-2b remains mostly unchanged at a variety of BSA to IFN alpha-2b protein ratios. A significant difference in alpha helix and beta sheet content was noted when the BSA to IFN alpha-2b ratio was 5:1 (w/w), suggesting a potential conformational change in IFN alpha-2b secondary structure when BSA is in molar excess.

Immunotherapeutic approaches for Alzheimer's disease in transgenic mouse models.

Alzheimer's disease (AD) is a member of a category of neurodegenerative diseases characterized by the conformational change of a normal protein into a pathological conformer with a high beta-sheet content that renders it resistant to degradation and neurotoxic. In the case of AD the normal soluble amyloid beta (sAbeta) peptide is converted into oligomeric/fibrillar Abeta. The oligomeric forms of Abeta are thought to be the most toxic, while fibrillar Abeta becomes deposited as amyloid plaques and congophilic angiopathy, which both serve as neuropathological markers of the disease. In addition, the accumulation of abnormally phosphorylated tau as soluble toxic oligomers and as neurofibrillary tangles is an essential part of the pathology. Many therapeutic interventions are under investigation to prevent and treat AD. The testing of these diverse approaches to ameliorate AD pathology has been made possible by the existence of numerous transgenic mouse models which each mirror different aspects of AD pathology. Perhaps the most exciting of these approaches is immunomodulation. Vaccination is currently being tried for a range of age associated CNS disorders with great success being reported in many transgenic mouse models. However, there is a discrepancy between these results and current human clinical trials which highlights the limitations of current models and also uncertainties in our understanding of the underlying pathogenesis of AD. No current AD Tg mouse model exactly reflects all aspects of the human disease. Since the underlying etiology of sporadic AD is unknown, the process of creating better Tg models is in constant evolution. This is an essential goal since it will be necessary to develop therapeutic approaches which will be highly effective in humans.

Silk nanospheres and microspheres from silk/pva blend films for drug delivery

Silk fibroin protein-based micro- and nanospheres provide new options for drug delivery due to their biocompatibility, biodegradability and their tunable drug loading and release properties. In the present study, we report a new aqueous-based preparation method for silk spheres with controllable sphere size and shape. The preparation was based on phase separation between silk fibroin and polyvinyl alcohol (PVA) at a weight ratio of 1/1 and 1/4. Water-insoluble silk spheres were easily obtained from the blend in a three step process: (1) air-drying the blend solution into a film, (2) film dissolution in water and (3) removal of residual PVA by subsequent centrifugation. In both cases, the spheres had approximately 30% beta-sheet content and less than 5% residual PVA. Spindle-shaped silk particles, as opposed to the spherical particles formed above, were obtained by stretching the blend films before dissolving in water. Compared to the 1/1 ratio sample, the silk spheres prepared from the 1/4 ratio sample showed a more homogeneous size distribution ranging from 300 nm up to 20 μm. Further studies showed that sphere size and polydispersity could be controlled either by changing the concentration of silk and PVA or by applying ultrasonication on the blend solution. Drug loading was achieved by mixing model drugs in the original silk solution. The distribution and loading efficiency of the drug molecules in silk spheres depended on their hydrophobicity and charge, resulting in different drug release profiles. The entire fabrication procedure could be completed within one day. The only chemical used in the preparation except water was PVA, an FDA-approved ingredient in drug formulations. Silk micro- and nanospheres reported have potential as drug delivery carriers in a variety of biomedical applications.

Dissection of a β‐barrel motif leads to a functional dimer: The case of the intestinal fatty acid binding protein

A lingering issue in the area of protein engineering is the optimal design of beta motifs. In this regard, the framework provided by intestinal fatty acid binding protein (IFABP) was successfully chosen to explore the consequences on structure and function of the redesign of natural motifs. A truncated form of IFABP (Delta 98 Delta) served to illustrate the nonintuitive notion that the integrity of the beta-barrel can indeed be compromised with no effect on the ability to attain a native-like fold. This is most likely the outcome of the key role played by the preservation of essential core residues. In the search for the minimal structural determinants of this fold, Delta 98 Delta offered room for further intervention. A dissection of this protein leads to a new abridged variant, Delta 78 Delta, containing 60% of the amino acids of IFABP. Spectroscopic analyses indicate that Delta 78 Delta retains substantial beta-sheet content and preserves tertiary interactions, displaying cooperative unfolding and binding activity. Most strikingly, this construct adopts a remarkably stable dimeric structure in solution. This phenomenon takes advantage of the inherent structural plasticity of this motif, likely profitting from edge-to-edge interactions between beta-sheets, whereas avoiding the most commonly occurring outcome represented by aggregation.

The relation between moisture-induced aggregation and structural changes in lyophilized insulin

Long-term stability is a critical factor in the successful development of protein pharmaceuticals. Due to the relative instability of proteins in aqueous solutions, they are formulated frequently and stored as lyophilized powders. Exposure of such powders to moisture constitutes a substantial storage problem leading to aggregation and inactivation. We have investigated the structural consequences of moisture sorption by lyophilized insulin under controlled humidity conditions by employing Fourier transform-infrared (FT-IR) microscopy. Lyophilized insulin samples were stored in humidity chambers under controlled conditions at 50(o)C. Protein aggregation studies were carried out by redissolving the insulin samples and measuring the amount of both soluble protein and insoluble aggregates. Near-UV circular dichroism spectra were collected to assess the tertiary structure. FT-IR microscopy studies were carried out to investigate secondary structural changes in solid-state insulin after incubation at different relative humidities. It was found that sorption of moisture was accompanied by small structural changes in lyophilized insulin at low levels of relative humidity (i.e. 11%). At higher relative humidity levels, structural changes were becoming more pronounced and were characterized by a loss in the alpha-helix and increase in beta-sheet content. The magnitude of the structural changes in tendency paralleled the solid-state instability data (i.e. formation of buffer-insoluble aggregates and loss in tertiary structure upon reconstitution). The results support the hypothesis that water sorption by lyophilized proteins enables structural transitions which can lead to protein aggregation and other deleterious phenomena.

Characterization of intrinsically disordered proteins with electrospray ionization mass spectrometry: Conformational heterogeneity of α‐synuclein

Conformational heterogeneity of alpha-synuclein was studied with electrospray ionization mass spectrometry by analyzing protein ion charge state distributions, where the extent of multiple charging reflects compactness of the protein conformations in solution. Although alpha-synuclein lacks a single well-defined structure under physiological conditions, it was found to sample four distinct conformational states, ranging from a highly structured one to a random coil. The compact highly structured state of alpha-synuclein is present across the entire range of conditions tested (pH ranging from 2.5 to 10, alcohol content from 0% to 60%), but is particularly abundant in acidic solutions. The only other protein state populated in acidic solutions is a partially folded intermediate state lacking stable tertiary structure. Another, more compact intermediate state is induced by significant amounts of ethanol used as a co-solvent and appears to represent a partially folded conformation with high beta-sheet content. Protein dimerization is observed throughout the entire range of conditions tested, although only acidic solutions favor formation of highly structured dimers of alpha-synuclein. These dimers are likely to present the earliest stages in protein aggregation leading to globular oligomers and, subsequently, protofibrils.

Backbone and side-chain 1H, 13C and 15N resonance assignments of LEN, a human immunoglobulin κIV light-chain variable domain

(1)H, (13)C and (15)N resonance assignments are presented for a recombinant 114 amino acid human immunoglobulin (Ig) kappaIV light-chain variable domain (VL) LEN, which displays a high degree of sequence identity with another human Ig kappaIV VL, SMA. While SMA is highly amyloidogenic in vivo and in vitro and has been linked to the pathogenesis of light-chain amyloidosis, LEN is non-amyloidogenic in vivo and can be converted to the amyloid state only in vitro under destabilizing conditions. Measurements of longitudinal and transverse amide (15)N relaxation rates confirm that, as expected, LEN is a dimer at physiological pH and typical concentrations used for NMR studies, and the analysis of secondary chemical shifts indicates that the protein has a high beta-sheet content. These findings are consistent with previously published biophysical data and the high-resolution X-ray structure of LEN.

Diversity of kinetic pathways in amyloid fibril formation

The kinetics of peptide oligomerization was investigated using Langevin Dynamics simulations and a coarse-grained peptide model. The simulations show a rich diversity of aggregation pathways, modulated by the beta-sheet propensity (flexibility) of the peptide. Aggregation into amyloidlike fibrils occurs via three main mechanisms: (i) formation of fibrils directly from the assembly of early ordered oligomers, (ii) fibril formation via the formation of on-pathway, nonfibrillar aggregates high in beta-sheet content, and (iii) formation of amorphous aggregates followed by reorganization to beta-sheet aggregates and to fibrils. beta-sheet, nonfibrillar aggregates also appeared as long-lived, "off-pathway" end-product species.

Purification and biochemical characterization of a novel α-glucosidase from Aspergillus niveus

An extracellular a-glucosidase produced by Aspergillus niveus was purified using DEAE-Fractogel ion-exchange chromatography and Sephacryl S-200 gel filtration. The purified protein migrated as a single band in 5% PAGE and 10% SDS-PAGE. The enzyme presented 29% of glycosylation, an isoelectric point of 6.8 and a molecular weight of 56 and 52 kDa as estimated by SDS-PAGE and Bio-Sil-Sec-400 gel filtration column, respectively. The enzyme showed typical alpha-glucosidase activity, hydrolyzing p-nitrophenyl alpha-D-glucopyranoside and presented an optimum temperature and pH of 65 degrees C and 6.0, respectively. In the absence of substrate the purified alpha-glucosidase was stable for 60 min at 60 degrees C, presenting t(50) of 90 min at 65 degrees C. Hydrolysis of polysaccharide substrates by alpha-glucosidase decreased in the order of glycogen, amylose, starch and amylopectin. Among malto-oligosaccharides the enzyme preferentially hydrolyzed malto-oligosaccharide (G10), maltopentaose, maltotetraose, maltotriose and maltose. Isomaltose, trehalose and beta-ciclodextrin were poor substrates, and sucrose and alpha-ciclodextrin were not hydrolyzed. After 2 h incubation, the products of starch hydrolysis measured by HPLC and thin layer chromatography showed only glucose. Mass spectrometry of tryptic peptides revealed peptide sequences similar to glucan 1,4-alpha-glucosidases from Aspergillus fumigatus, and Hypocrea jecorina. Analysis of the circular dichroism spectrum predicted an a-helical content of 31% and a beta-sheet content of 16%, which is in agreement with values derived from analysis of the crystal structure of the H. jecorina enzyme.

Structural and Functional Alterations of Catalase Induced by Acriflavine, a Compound Causing Apoptosis and Necrosis

Acriflavine is an antiseptic agent causing both apoptosis and necrosis in yeast. In this work, its effect on the structure and function of catalase, a vital enzyme actively involved in protection against oxidative stress, was investigated. In vitro kinetic studies showed that acriflavine inhibited the enzymatic activity in a competitive manner. The residual activity detectable after preincubation of catalase (1.5 nmol/L) with various concentrations of acriflavine went from 50% to 20% of the control value as the acriflavine concentration increased from 30 to 90 micromol/L. Correlatively with the decrease in activity, alterations in the enzyme's conformation were observed as indicated by fluorescence spectroscopy, circular dichroism spectroscopy, and electronic absorption spectroscopy. The enzyme's intrinsic fluorescence obtained upon excitation at either 297 nm (tryptophan residues) or 280 nm (tyrosine and tryptophan residues) decreased as a function of acriflavine concentration. Circular dichroism studies showed alterations of the protein structure by acriflavine with up to 13% decrease in alpha helix, 16% increase in beta-sheet content, 17% increase in random coil, and 4% increase in beta turns. Spectrophotometric studies showed a blueshift and modifications in the chromicity of catalase at 405 nm, corresponding to an absorbance band due to the enzyme's prosthetic group. Thus, acriflavine induced in vitro a profound change in the structure of catalase so that the enzyme could no longer function. Our results showed that acriflavine, a compound producing apoptosis and necrosis, can have a direct effect on vital functions in cells by disabling key enzymes.

Manganese-induced changes of the biochemical characteristics of the recombinant wild-type and mutant PrPs

Manganese may play some roles in the pathogenesis of prion diseases. In this study, recombinant human wild-type (WT) PrP and PrP mutants with deleted or inserted octarepeats were exposed to manganese, and their biochemical and biophysical characteristics were evaluated by proteinase K (PK) digestion, sedimentation experiments, transmission electron microscopy and circular dichroism. It demonstrated that incubation of manganese remarkably increased PK-resistances, protein aggregations and beta-sheet contents of the PrPs. Moreover, the PrP mutants of inserted or deleted octarepeats were much vulnerable to the influence of manganese, which showed obviously more aggregation and higher beta-sheet content than that of WT-PrP. It highlights that the effect of manganese on the PrP seems to lie on the incorrectness of the octarepeats numbers. The association of the octarepeats number of PrP with manganese may further provide insight into the unresolved biological function of PrP in the neurons.

Enhancement of beta-sheet assembly by cooperative hydrogen bonds potential

The roughness of energy landscapes is a major obstacle to protein structure prediction, since it forces conformational searches to spend much time struggling to escape numerous traps. Specifically, beta-sheet formation is prone to stray, since many possible combinations of hydrogen bonds are dead ends in terms of beta-sheet assembly. It has been shown that cooperative terms for backbone hydrogen bonds ease this problem by augmenting hydrogen bond patterns that are consistent with beta sheets. Here, we present a novel cooperative hydrogen-bond term that is both effective in promoting beta sheets and computationally efficient. In addition, the new term is differentiable and operates on all-atom protein models. Energy optimization of poly-alanine chains under the new term led to significantly more beta-sheet content than optimization under a non-cooperative term. Furthermore, the optimized structure included very few non-native patterns. The new term is implemented within the MESHI package and is freely available at http://cs.bgu.ac.il/ approximately meshi.

Functional and structural alterations induced by copper in xanthine oxidase

Xanthine oxidase (XO), a key enzyme in purine metabolism, produces reactive oxygen species causing vascular injuries and chronic heart failure. Here, copper's ability to alter XO activity and structure was investigated in vitro after pre-incubation of the enzyme with increasing Cu(2+) concentrations for various periods of time. The enzymatic activity was measured by following XO-catalyzed xanthine oxidation to uric acid under steady-state kinetics conditions. Structural alterations were assessed by electronic absorption, fluorescence, and circular dichroism spectroscopy. Results showed that Cu(2+) either stimulated or inhibited XO activity, depending on metal concentration and pre-incubation length, the latter also determining the inhibition type. Cu(2+)-XO complex formation was characterized by modifications in XO electronic absorption bands, intrinsic fluorescence, and alpha-helical and beta-sheet content. Apparent dissociation constant values implied high- and low-affinity Cu(2+) binding sites in the vicinity of the enzyme's reactive centers. Data indicated that Cu(2+) binding to high-affinity sites caused alterations around XO molybdenum and flavin adenine dinucleotide centers, changes in secondary structure, and moderate activity inhibition; binding to low affinity sites caused alterations around all XO reactive centers including FeS, changes in tertiary structure as reflected by alterations in spectral properties, and drastic activity inhibition. Stimulation was attributed to transient stabilization of XO optimal conformation. Results also emphasized the potential role of copper in the regulation of XO activity stemming from its binding properties.

Surface Properties and Conformation of Nephila clavipes Spider Recombinant Silk Proteins at the Air−Water Interface

The dragline fiber of spiders is composed of two proteins, the major ampullate spidroins I and II (MaSpI and MaSpII). To better understand the assembly mechanism and the properties of these proteins, the adsorption behavior of the recombinant proteins of the spider Nephila clavipes produced by Nexia Biotechnologies Inc. has been studied at the air-water interface using ellipsometry, surface pressure, rheological, and infrared measurements. The results show that the adsorption is more rapid and more molecules are present at the interface for MaSpII than for MaSpI. MaSpII has thus a higher affinity for the interface than MaSpI, which is consistent with its higher aggregation propensity in water. The films formed at the interface consist of networks containing a high content of intermolecular beta-sheets as revealed by the in situ polarization modulation infrared absorption reflection spectra. The infrared results further demonstrate that, for MaSpI, the beta-sheets are formed as soon as the proteins adsorb to the interface while for MaSpII the beta-sheet formation occurs more slowly. The amount of beta-sheets is lower for MaSpII than for MaSpI, most likely due to the presence of proline residues in its sequence. Both proteins form elastic films, but they are heterogeneous for MaSpI and homogeneous for MaSpII most probably as a result of a more ordered and slower aggregation process for MaSpII. This difference in their mechanism of assembly and interfacial behaviors does not seem to arise from their overall hydrophobicity or from a specific pattern of hydrophobicity, but rather from the longer polyalanine motifs, lower glycine content, and higher proline content of MaSpII. The propensity of both spidroins to form beta-sheets, especially the polyalanine blocks, suggests the participation of both proteins in the silk's beta-sheet crystallites.

Electrochemical and Conformational Consequences of Copper (CuI and CuII) Binding to β‐Amyloid(1–40)

Copper-induced structural rearrangements of Abeta40 structure and its redox properties are described in this study. Electrochemical and fluorescent methods are used to characterise the behaviour of Abeta-Cu species. The data suggest that time-dependent folding of Abeta-Cu species may cause changes in the redox potentials.Extracellular deposits of beta-amyloid (Abeta) into senile plaques are the major features observed in brains of Alzheimer's disease (AD) patients. A high concentration of copper has been associated with insoluble amyloid plaques. It is known that Abeta(1-40) can bind copper with high affinity, but electrochemical properties of Abeta(1-40)-Cu complexes are not well-characterised. In this study we demonstrate that complexation of copper (both as Cu(I) and Cu(II)) by Abeta(1-40) reduces the metal electrochemical activity. Formation of copper-Abeta(1-40) complexes is associated with alteration of the redox potential. The data reveal significant redox activity of fresh Abeta-copper solutions. However, copper-induced structural rearrangements of the peptide, documented by CD, correspond with time-dependent changes of formal reduction potentials (E(0')) of the complex. Fluorescent and electrochemical (cyclic voltammetry and differential pulse voltammetry) techniques suggest that reduction of the redox activity by Abeta-Cu complexes could be attributed to conformational changes that diminished copper accessibility to the external environment. According to our evidence, conformational rearrangements, induced by copper binding to amyloid, elongate the time necessary to attain the same beta-sheet content as for the metal-free peptide. Although the redox activity of Abeta-Cu complexes diminishes in a time-dependent manner, they are not completely devoid of toxicity as they destabilize red blood cells osmotic fragility, even after prolonged incubation.

Solid-State NMR Studies of Gas Vesicle Structure

Gas vesicles are gas-filled organelles that allow algae, bacteria and archaea to adjust their position in the water column for optimal illumination and aeration. The spindle-shaped vesicles are typically ∼500 nm long and ∼75 nm wide, with ∼1.9 nm thick walls and their shells consist exclusively of protein, primarily the highly hydrophobic GvpA monomer (70 residues), with a permeability such that the vesicle is filled with gas of atmospheric composition. Electron microscopy has shown that the GvpA monomers are arranged in a low-pitched helix; infrared spectroscopy shows considerable beta-sheet content, in agreement with results from X-ray scattering; and atomic force microscopy shows beta-strands tilted at an angle relative to the vesicle axis that is consistent with X-ray scattering measurements on partially aligned vesicles. To gain further insight into the molecular structure and interactions that grant gas vesicles their remarkable physical properties, atomic resolution data is required. However, insolubility prevents the use of solution NMR or crystallography, and multiple scattering frustrates high-resolution electron microscopy. Here, we present the results of solid-state NMR experiments aimed at characterizing the structure of GvpA in intact, deflated gas vesicles from Anabaena flos-aquae. Fairly complete dipolar correlation spectra, indicating a largely rigid and well-ordered system, have allowed resonance assignments for ∼80% of the protein sequence. These chemical shifts provide evidence for the presence of both beta-strand and alpha-helix elements in the GvpA backbone. Furthermore, certain regions of the sequence present duplicated resonances, which suggest that the basic structural subunit of gas vesicles is an asymmetric GvpA dimer. Finally, molecular mobility and preliminary tertiary structural characteristics are also discussed.

Generic Coarse-Grained Model for Protein Folding and Aggregation

The complexity involved in protein structure is not only due to the rich variety of amino acids, consistent with the random heteropolymer picture, but also the weak interactions involved, comparable to thermal energy, and important cooperative phenomena. This presents a challenge in computer simulations, as it is associated with high-dimensionality and ruggedness of the free energy landscape as well as long equilibration times, frequently exceeding what can be handled in atomistic studies. We have recently developed a coarse-grained (CG) implicit solvent peptide model which has been designed to reproduce key consequences of the abovementioned weak interactions. Its intermediate level of resolution, four beads per amino acid, allows for accurate sampling of local conformations, in particular secondary structure, by designing a force field that relies on simple interactions (e.g. hydrogen bonds, hydrophobicity). A realistic ratio of alpha-helix to beta-sheet content is achieved by mimicking a nearest-neighbor dipolar interaction. In the present study, we tune the model in order to fold helical proteins while systematically comparing the structure with NMR data. Very good agreement is achieved for proteins that have simple tertiary structures, which implies that the force field is able to reproduce important cooperativity features between amino acids. We further probe these effects by looking at peptide aggregation scenarios. Hydrophobic peptide fragments cooperatively form largescale beta-sheet structures. The model is able to reproduce features from atomistic simulations on a qualitative basis. The large-scale and long-term regime that this CG model offers, coupled with our design criteria (folding and realistic alpha/beta content), make it very suitable for many biological processes, such as misfolding and oligomerization involved in neurodegenerative diseases.

A hypoallergenic variant of Der p 1 as a candidate for mite allergy vaccines

Recombinant hypoallergens that display reduced allergenicity but retain T-cell reactivity represent promising candidates to improve the safety and efficacy of allergen-specific vaccines or immunotherapy. The current study reports the immunologic characterization of a hypoallergenic variant of the major mite allergen Der p 1. The recombinant proform of Der p 1 (ProDer p 1) was expressed in Escherichia coli (ProDer p 1 coli), purified and characterized at the level of its secondary structure, and IgE and T-cell reactivities. Moreover, the prophylactic potential of ProDer p 1 coli vaccinations was evaluated in a murine Der p 1 sensitization model. After purification and refolding, ProDer p 1 coli remained aggregated with a higher beta-sheet content and altered Der p 1 conformational epitopes compared with the correctly folded monomeric ProDer p 1 produced in Chinese hamster ovary cells. Both ProDer p 1 forms were able to retain the Der p 1-specific T-cell reactivity but direct ELISA, competitive inhibition, and rat basophil leukemia assays clearly showed that ProDer p 1 coli displays a very weak IgE reactivity. Mice vaccinations with aggregated ProDer p 1 adjuvanted with alum induced a T(H)1-biased immune response that prevented the subsequent allergic response after Der p 1 sensitization and airway challenge with aerosolized mite extracts. Furthermore, ProDer p 1 coli treatment inhibited the development of airway eosinophilia and airway hyperresponsiveness to inhaled methacholine. Aggregated forms of Der p 1 could represent hypoallergens suitable for the prevention of mite allergy.

Can copper binding to the prion protein generate a misfolded form of the protein?

The native prion protein (PrP) has a two domain structure, with a globular folded alpha-helical C-terminal domain and a flexible extended N-terminal region. The latter can selectively bind Cu(2+) via four His residues in the octarepeat (OR) region, as well as two sites (His96 and His111) outside this region. In the disease state, the folded C-terminal domain of PrP undergoes a conformational change, forming amorphous aggregates high in beta-sheet content. Cu(2+) bound to the ORs can be redox active and has been shown to induce cleavage within the OR region, a process requiring conserved Trp residues. Using computational modeling, we have observed that electron transfer from Trp residues to copper can be favorable. These models also reveal that an indole-based radical cation or Cu(+) can initiate reactions leading to protein backbone cleavage. We have also demonstrated, by molecular dynamics simulations, that Cu(2+) binding to the His96 and His111 residues in the remaining PrP N-terminal fragment can induce localized beta-sheet structure, allowing us to suggest a potential mechanism for the initiation of beta-sheet misfolding in the C-terminal domain by Cu(2+).