Mechanical, microstructural and solubility properties of pectin/poly(vinyl alcohol) blends

Citrus pectin was blended and cast into films with poly(vinyl alcohol) (PVOH). PVOH and pectin were miscible in all proportions. Dynamic mechanical analysis revealed that pectin controls exhibited no thermal transitions, whereas PVOH controls exhibited a glass transition temperature (T_g) over a broad temperature range commencing at about 0 °C and ending about 50 °C. A mixture of 49% pectin, 21% PVOH and 30% glycerol exhibited lower storage moduli and more flexibility than comparable mixtures of either pectin/PVOH or pectin/glycerol. Scanning electron microscopy and phase contrast optical microscopy indicated that the mixture was biphasic and a compatible composite either of PVOH in pectin or pectin in PVOH depending on which macromolecule was in excess. Elongation to break measurements revealed that pectin/PVOH films underwent a brittle to ductile transition with increasing PVOH composition. The addition of glycerol to pectin/PVOH films increased ductility significantly when films were relatively brittle. Initial moduli (IM) as a function of composition gave complex curves which exhibited either one or two local maxima depending on such factors as degree of hydrolysis and molar mass of the PVOH in addition to the moisture content of the film. Solubility studies in water revealed that, at 30 and 50 °C, only films with 30% PVOH or less were soluble. At 70 °C, all compositions were soluble but films containing pectin dissolved more rapidly than those without. The solution kinetics of pectin/PVOH films with 30% or less PVOH were approximated with zero-order kinetics and activation energies were about 3–5 kcal mol⁻¹. In general, addition of PVOH to pectin films resulted in films with more PVOH-like properties and addition of pectin to PVOH films resulted in films with more pectin-like properties.     

Amyloid fibrils as a nanoscaffold for enzyme immobilization

Amyloid fibrils are a misfolded state, formed by many proteins when subjected to denaturing conditions. Their constituent amino acids make them ideally suited as a readily functionalized nanoscaffold for enzyme immobilization and their strength, stability, and nanometer size are attractive features for exploitation in the creation of new bionanomaterials. We report successful functionalization of amyloid fibrils by conjugation to glucose oxidase (GOD) using glutaraldehyde. GOD retained activity upon attachment and successful cross‐linking was determined using electrophoresis, centrifugation, sucrose gradient centrifugation, and TEM. The resulting functionalized enzyme scaffold was then incorporated into a model poly(vinyl alcohol) (PVOH) film, to create a new bionanomaterial. The antibacterial effect of the functionalized film was then tested on E. coli, the growth of which was inhibited, demonstrating the incorporation of GOD antibacterial activity into the PVOH film. The incorporation of the GOD‐functionalized amyloid fibrils into PVOH provides an excellent ‘proof of concept’ model for the creation of a new bionanomaterial using a functionalized amyloid fibril scaffold. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Quercetin inhibits amyloid fibrillation of bovine insulin and destabilizes preformed fibrils

Growing interest and research efforts have recently been focused on elucidating the molecular mechanism of amyloid formation and the screening of effective inhibitors to interrupt amyloid structures. In the present study, the anti-amyloidogenic effects of quercetin were investigated in vitro using bovine insulin as a model protein. The results demonstrated that quercetin dose-dependently inhibited amyloid formation of insulin. Moreover, quercetin destabilized the preformed insulin fibrils and transformed the fibrils into amorphous aggregates. Hemolysis was observed when human erythrocytes were co-incubated with insulin fibrils. Quercetin inhibited fibril-induced hemolysis in a dose-dependent manner. SDS–PAGE showed that insulin fibrils induced the aggregation of cytoskeletal proteins of erythrocyte membranes and that quercetin attenuated this fibril-induced cytoskeletal aggregation. The results of the present work suggest that quercetin may serve as a lead structure for the design of novel anti-amyloidogenic drugs.     

Controlling the dimensions of amyloid fibrils: toward homogenous components for bionanotechnology

Amyloid fibrils have been recognized as having potential in a variety of bionanotechnological applications. However, realization of these applications is constrained by a lack of control over morphology and alignment, both crucial for potential end uses. This article focuses on the use of growth and storage conditions to control the length of amyloid fibrils formed from bovine insulin, with length distributions constructed from transmission electron microscopy (TEM) images. Growth temperature, pH, protein concentration, and storage conditions were examined and were seen to offer a range of conditions that favor different length distribution. The use of amyloid fibrils as nanowires is one area where control of fibril dimensions is desirable, for experimental setup and endpoint applications. The conductive properties of fibrils formed from bovine insulin are presented, with these insulin fibrils being shown to have high resistivity in their unmodified state, with current values in the nanoamp range. These low current values can be increased via modification, or the fibrils used in their native state in applications where low current values are desirable. These findings, coupled with the ability to predict and select for various insulin amyloid fibril dimensions, enhances their utility as nanomaterials.     

The binding of thioflavin-T to amyloid fibrils: localisation and implications

Amyloid fibrils are a polymeric form of protein, involving a continuous beta-sheet with the strands perpendicular to the long axis of the fibril. Although typically implicated in diseases such as Alzheimer's disease and the transmissible spongiform encephalopathies, non disease-associated protein can also be converted into amyloid fibrils. Traditionally, amyloid fibrils are identified via the use of specific dyes such as Congo red and thioflavin-T, although their specificity is ill understood. Recently, solutions of bovine insulin and bovine beta-lactoglobulin have been found to form spherulites, micron-sized spherical structures containing radially arranged amyloid fibrils. When studied by confocal microscopy using polarised laser light and thioflavin-T, a consistent pattern of emission, rather than a uniform disc, was observed. This suggests the dye binds in a specific, regular fashion to amyloid fibrils. Confocal microscopy studies of thioflavin-T aligned in stretched poly-vinyl alcohol films showed that the dye dipole excitation axis lies parallel to the long molecular axis. Therefore, thioflavin-T binds to amyloid fibrils such that their long axes are parallel. We propose binding occurs in 'channels' that run along the length of the beta-sheet. Steric interactions between dye molecules and side chains indicate why thioflavin-T fluoresces more intensely when bound to amyloid fibrils and can explain why this interaction with amyloid fibrils is specific, but with varying efficiency.     

Multistep Changes in Amyloid Structure Induced by Cross-Seeding on a Rugged Energy Landscape

Amyloid fibrils are aberrant protein aggregates associated with various amyloidoses and neurodegenerative diseases. It is recently indicated that structural diversity of amyloid fibrils often results in different pathological phenotypes, including cytotoxicity and infectivity. The diverse structures are predicted to propagate by seed-dependent growth, which is one of the characteristic properties of amyloid fibrils. However, much remains unknown regarding how exactly the amyloid structures are inherited to subsequent generations by seeding reaction. Here, we investigated the behaviors of self- and cross-seeding of amyloid fibrils of human and bovine insulin in terms of thioflavin T fluorescence, morphology, secondary structure, and iodine staining. Insulin amyloid fibrils exhibited different structures, depending on species, each of which replicated in self-seeding. In contrast, gradual structural changes were observed in cross-seeding, and a new type of amyloid structure with distinct morphology and cytotoxicity was formed when human insulin was seeded with bovine insulin seeds. Remarkably, iodine staining tracked changes in amyloid structure sensitively, and singular value decomposition analysis of the ultraviolet-visible absorption spectra of the fibril-bound iodine has revealed the presence of one or more intermediate metastable states during the structural changes. From these findings, we propose a propagation scheme with multistep structural changes in cross-seeding between two heterologous proteins, which is accounted for as a consequence of the rugged energy landscape of amyloid formation.     

Chemical modification of insulin in amyloid fibrils

We have investigated the chemical modification of insulin under conditions that promote the conversion of the soluble protein into amyloid fibrils. The modifications that are incorporated into the fibrils include deamidation of Asn A21, Asn B3, and Gln B4. In order to prepare fibrils with minimal deamidation of these residues, the kinetics of aggregation were accelerated by seeding with aliquots of a solution containing preformed fibrils. The resulting fibrils were then reincubated to determine the extent to which chemical modification occurs in the fibril itself. The deamidation of Asn A21 in particular could be followed in detail. Deamidation of this residue in the fibrillar form of insulin was found to occur in only 52 +/- 5% of molecules. This result indicates that there are at least two different packing environments of insulin molecules in the fibrils and suggests that the characterization of chemical modifications may be a useful probe of the environment of polypeptide chains within amyloid fibrils.     

Insulin amyloid fibrillation at above 100 degrees C: new insights into protein folding under extreme temperatures

To investigate the folding behavior of amyloidogenic proteins under extreme temperatures, the kinetics of fibrillation and accompanying secondary structure transitions of bovine insulin were studied for temperatures ranging up to 140 degrees C. The presence of extreme heat stress had traditionally been associated with irreversible denaturation of protein while the initial steps of such a denaturation process may be common with a fibril formation pathway of amyloidogenic proteins. The present work demonstrates the ability of insulin to form amyloid fibrils at above 100 degrees C. Amyloid formation was gradually replaced by random coil generation after approximately 80 degrees C until no amyloid was detected at 140 degrees C. The morphology of insulin amyloid fibrils underwent sharp changes with increasing the temperature. The dependence of amyloid formation rate on incubation temperature followed non-Arrhenius kinetics, which is explained by temperature-dependent enthalpy change for amyloid formation. The intermediate stage of amyloid formation and random coil generation consisted of a partially folded intermediate common to both pathways. The fully unfolded monomers in random coil conformation showed partial reversibility through this intermediate by reverting back to the amyloid pathway when formed at 140 degrees C and incubated at 100 degrees C. This study highlights the non-Arrhenius kinetics of amyloid fibrillation under extreme temperatures, and elucidates its intermediate stage common with random coil formation.     

Bovine Insulin Filaments Induced by Reducing Disulfide Bonds Show a Different Morphology, Secondary Structure, and Cell Toxicity from Intact Insulin Amyloid Fibrils

Amyloid fibrils are associated with more than 20 diseases, including Alzheimer's disease and type II diabetes. Insulin is a 51-residue polypeptide hormone, with its two polypeptide chains linked by one intrachain and two interchain disulfide bonds, and has long been known to self-assemble in vitro into amyloid fibrils. We demonstrate here that bovine insulin forms flexible filaments in the presence of a reducing agent, Tris (2-carboxyethyl) phosphine. The insulin filaments, possibly formed due to partial reduction of S-S bonds in insulin molecules, differ from intact insulin fibrils in terms of their secondary structure. The insulin filaments were determined to have an antiparallel β-sheet structure, whereas the insulin fibrils have a parallel β-sheet structure. Of importance, the cell toxicity of the insulin filaments was remarkably lower than that of the insulin fibrils. This finding supports the idea that cell toxicity of amyloids correlates with their morphology. The remarkably low toxicity of the filamentous structure should shed new light on possible pharmacological approaches to the various diseases caused by amyloid fibrils.     

Asymmetric amyloid fibril elongation: a new perspective on a symmetric world

Amyloids are insoluble, fibrous proteins formed through the aggregation of misfolded proteins. They accumulate in the tissue of individuals with degenerative diseases, such as Parkinson's and Alzheimer's. The purpose of this study was to determine whether fibril growth from an initial model fibril seed is unidirectional or bidirectional. The prevailing theory on amyloid formation is that a symmetric fibril elongates equally from both ends. This study provides evidence to the contrary; the process occurs predominately unidirectionally, demonstrating that amyloid fibrils may be asymmetric and propagate mostly in one direction. Alexa Fluor 568 labeled insulin fibrils were seeded into a native insulin solution and allowed to elongate at 65°C while the kinetics of fibril growth was monitored. The resulting elongated fibrils were labeled with thioflavin-T, and the fluorescent images of the fibrils show that a majority of the elongated fibrils propagated along only one end of the seed, with the remaining labeled fibrils having bidirectional elongation or no elongation. Using a crystallographic model, we offer a structural explanation for asymmetric growth of the insulin fibrils. Thus, instead of the current view that fibrils grow symmetrically from both ends of the fibril, this is the first evidence that insulin amyloid fibrils formed in solution are asymmetric and appear to grow from only one end.     

Immobilization of organophosphate hydrolase on an amyloid fibril nanoscaffold: towards bioremediation and chemical detoxification

Organophosphate hydrolase has potential as a bioremediation and chemical detoxification enzyme, but the problems of reusability and stability need to be addressed to use this enzyme on an industrial scale. Immobilizing the enzyme to a nanoscaffold may help to solve these problems. Amyloid fibrils generated from insulin and crystallin provided a novel nanoscaffold for the immobilization of organophosphate hydrolase, using glutaraldehyde as the crosslinking reagent. Electrophoretic, centrifugation, and temperature stability experiments, together with transmission electron microscopy were undertaken to verify that crosslinking had successfully occurred. The resulting fibrils remained active towards the substrate paraoxon and when immobilized to the insulin amyloid fibrils, the enzyme exhibited a significant (～ 300%) increase in the relative temperature stability at 40, 45, and 50°C (as measured by comparing the initial enzyme activity to the activity remaining after heating), compared to free enzyme. This confirms that amyloid fibrils could provide a new type of nanoscaffold for enzyme immobilization.     

Fluorescence quantum yield of thioflavin T in rigid isotropic solution and incorporated into the amyloid fibrils

In this work, the fluorescence of thioflavin T (ThT) was studied in a wide range of viscosity and temperature. It was shown that ThT fluorescence quantum yield varies from 0.0001 in water at room temperature to 0.28 in rigid isotropic solution (T/η→0). The deviation of the fluorescence quantum yield from unity in rigid isotropic solution suggests that fluorescence quantum yield depends not only on the ultra-fast oscillation of ThT fragments relative to each other in an excited state as was suggested earlier, but also depends on the molecular configuration in the ground state. This means that the fluorescence quantum yield of the dye incorporated into amyloid fibrils must depend on its conformation, which, in turn, depends on the ThT environment. Therefore, the fluorescence quantum yield of ThT incorporated into amyloid fibrils can differ from that in the rigid isotropic solution. In particular, the fluorescence quantum yield of ThT incorporated into insulin fibrils was determined to be 0.43. Consequently, the ThT fluorescence quantum yield could be used to characterize the peculiarities of the fibrillar structure, which opens some new possibilities in the ThT use for structural characterization of the amyloid fibrils.     

The effect of exposing a critical hydrophobic patch on amyloidogenicity and fibril structure of insulin

It is widely accepted that the formation of amyloid fibrils is one of the natural properties of proteins. The amyloid formation process is associated with a variety of factors, among which the hydrophobic residues play a critical role. In this study, insulin was used as a model to investigate the effect of exposing a critical hydrophobic patch on amyloidogenicity and fibril structure of insulin. Porcine insulin was digested with trypsin to obtain desoctapeptide-(B23–B30) insulin (DOI), whose hydrophilic C-terminal of B-chain was removed and hydrophobic core was exposed. The results showed that DOI, of which the ordered structure (predominantly α-helix) was markedly decreased, was more prone to aggregate than intact insulin. As to the secondary structure of amyloid fibrils, DOI fibrils were similar to insulin fibrils formed under acidic condition, whereas under neutral condition, insulin formed less polymerized aggregates by showing decreased β-sheet contents in fibrils. Further investigation on membrane damage and hemolysis showed that DOI fibrils induced significantly less membrane damage and less hemolysis of erythrocytes compared with those of insulin fibrils. In conclusion, exposing the hydrophobic core of insulin can induce the increase of amyloidogenicity and formation of higher-order polymerized fibrils, which is less toxic to membranes.     

Investigation of the kinetics of insulin amyloid fibrils formation

Today, the investigation of the structure of ordered protein aggregates-amyloid fibrils, the influence of the native structure of the protein and the external conditions on the process of fibrillation-is the subject of intense investigations. The aim of the present work is to study the kinetics of formation of insulin amyloid fibrils at low pH values (conditions that are used at many stages of the isolation and purification of the protein) using the fluorescent probe thioflavin T. It is shown that the increase of the fluorescence intensity of ThT during the formation of amyloid fibrils is described by a sigmoidal curve, in which three areas can be distinguished: the lag phase, growth, and a plateau, which characterize the various stages of fibril formation. Despite the variation in the length of the lag phase at the same experimental conditions (pH and temperature), it is seen to drop during solution stirring and seeding. Data obtained by electron microscopy showed that the formed fibrils are long, linear filaments ～20 nm in diameter. With increasing incubation time, the fibril diameter does not change, while the length increases to 2–3 μm, which is accompanied by a significant increase in the number of fibril aggregates. All the experimental data show that, irrespective of the kinetics of formation of amyloid fibrils, their properties after the completion of the fibrillation process are identical. The results of this work, together with the previous studies of insulin amyloid fibrils, may be important for clarification the mechanism of their formation, as well as for the treatment of amyloidosis associated with the aggregation of insulin.     

Amyloid fibrillation and cytotoxicity of insulin are inhibited by the amphiphilic surfactants

Amyloid fibrils have been associated with at least 25 different degenerative diseases. The 51-residue polypeptide hormone insulin, which is associated with type II diabetes, has been shown to self-assemble to form amyloid fibrils in vitro. With bovine insulin as a model, the research presented here explores the effects of two amphiphilic surfactants (1,2-dihexanoyl-sn-glycero-3-phosphocholine (di-C7-PC) and 1,2-diheptanoyl-sn-glycero-3-phosphocholine (di-C7-PC)) on the in vitro fibrillation process of bovine insulin at pH 2.0 and 55 °C. We demonstrated that insulin fibrillation may be inhibited by both surfactants in a dose-dependent fashion. The best inhibition of fibril formation is observed when insulin is incubated with 4 mM di-C7-PC. Moreover, the addition of either surfactant at the concentrations studied attenuated insulin fibril-induced cytotoxicity in both PC12 and SH-SY5Y cell lines. The results from this work may contribute to the understanding of the molecular factors affecting amyloid fibrillation and the molecular mechanism(s) of the interactions between the membrane and amyloid proteins.     

Amyloid fibril formation of alpha-synuclein is accelerated by preformed amyloid seeds of other proteins: implications for the mechanism of transmissible conformational diseases

Alpha-synuclein is one of the causative proteins of familial Parkinson disease, which is characterized by neuronal inclusions named Lewy bodies. Lewy bodies include not only alpha-synuclein but also aggregates of other proteins. This fact raises a question as to whether the formation of alpha-synuclein amyloid fibrils in Lewy bodies may occur via interaction with fibrils derived from different proteins. To probe this hypothesis, we investigated in vitro fibril formation of human alpha-synuclein in the presence of preformed fibril seeds of various different proteins. We used three proteins, Escherichia coli chaperonin GroES, hen lysozyme, and bovine insulin, all of which have been shown to form amyloid fibrils. Very surprisingly, the formation of alpha-synuclein amyloid fibril was accelerated markedly in the presence of preformed seeds of GroES, lysozyme, and insulin fibrils. The structural characteristics of the natively unfolded state of alpha-synuclein may allow binding to various protein particles, which in turn triggers the formation (extension) of alpha-synuclein amyloid fibrils. This finding is very important for understanding the molecular mechanism of Parkinson disease and also provides interesting implications into the mechanism of transmissible conformational diseases.     

On the use of size exclusion chromatography for the resolution of mixed amyloid aggregate distributions: I. equilibrium partition models

In this study, we investigated the theoretical potential of size exclusion chromatography (SEC) for resolving mixtures of protein aggregates (of various sizes and shapes) produced in the generation of amyloid fibrils. We present our findings in the form of an equilibrium partition model. We first review the general characteristics of SEC and discuss the physicochemical features affecting solute transport and partition. We then develop new methods for estimating the transport and partition coefficients of protein aggregates on the basis of their molecular dimensions and the SEC column properties. We detail how these calculated properties can be used to estimate the likely resolving power of an SEC column. Model predictions were found to be in general agreement with experimental data gained from the measurement of the elution profile of sheared amyloid fibrils prepared from bovine insulin and passed through a Superose 6 precision SEC column. Our formalism should provide a basic appreciation of the competing factors at work and allow an informed choice to be made for optimal selection of SEC column medium to separate a desired size range of aggregate.     

Independent heterologous fibrillation of insulin and its B-chain peptide

Insulin is very prone to form amyloid fibrils under slightly destabilizing conditions, and the B-chain region plays a critical role in the fibrillation. We show here that the isolated B-chain peptide of bovine insulin also forms fibrils at both acidic and neutral pH. When a mixture of insulin and the B-chain peptide was incubated at either acidic or neutral pH, the formation of fibrils was clearly separated into two phases, with the faster phase corresponding to the formation of homogeneous fibrils from the B-chain and the slower phase corresponding to homogeneous fibrillation of insulin. To further investigate the interaction (or lack thereof) between the two polypeptides, we examined the effects of cross-seeding. The results indicate that seeds of B-chain fibrils accelerate the fibrillation of insulin at pH 1.6 and inhibit the fibrillation at pH 7.5, but seeds of insulin fibrils have little effect on the fibrillation of the B-chain. We conclude that at pH 7.5 simultaneous independent homologous fibrillation occurs, but at low pH, heterologous fibrillation takes place, and with B-chain seeding of insulin, a unique conformation of fibrils is formed. Our results demonstrate that in the co-aggregation of closely related peptides each peptide species may undergo concurrent homogeneous or heterologous polymerization and that fibrils of one species may or may not seed fibrillation of the other. The results demonstrate the significant "species" barrier in amyloid fibril formation between fibrillation induced by different fibrils. A model for the fibrillation of the heterogeneous system of insulin and B-chain insulin is proposed.     

CD studies on films of amyloid proteins and polypeptides: quantitative g-factor analysis indicates a common folding motif

Irrespective of the constituent protein, all amyloid fibrils show similar morphology in the electron microscope and x-ray diffraction patterns characteristic of a "cross-beta" structure, with extended beta-strands perpendicular to the fibril's long axis. Little is known about the amount or type of this structure. I have measured CD spectra of films formed from a number of amyloid proteins and polypeptides, and estimated their contents of extended secondary structure, by analysis of their g-factor spectra, the ratio of the CD and absorbance signals (P. McPhie, Analytical Biochemistry, 2001, Vol. 293, pp. 109-119). Amyloid films of Abeta-(1-40) peptide, beta-2-microglobulin, insulin, and three homopolypeptides show very intense CD spectra, compatible with the presence of a beta-helix-like structure, arranged in a common framework in the fibrils. The extent of this structure was estimated as 45-80% in the protein fibrils and 30-80% in the polypeptide fibrils.     

Highly Amyloidogenic Two-chain Peptide Fragments Are Released upon Partial Digestion of Insulin with Pepsin

Proteases play a well recognized role in the emergence of highly aggregation-prone protein fragments in vivo, whereas in vitro limited proteolysis is often employed to probe different phases of amyloidogenic pathways. Here, we show that addition of moderate amounts of pepsin to acidified bovine insulin at close to physiological temperature results in an abrupt self-assembly of amyloid-like fibrils from partially digested insulin fragments. Biochemical analysis of the pepsin-induced fibrils implicates peptide fragments (named H) consisting of the 13 or 15 N-terminal residues of the A-chain and 11 or 13 N-terminal residues of the B-chain linked by the disulfide bond between Cys-7A–Cys-7B as the main constituents. There are up to eight pepsin-cleavage sites remaining within the double chain peptide, which become protected upon fast fibrillation unless concentration of the enzyme is increased resulting in complete digestion of insulin. Controlled re-association of H-peptides leads to “explosive” fibrillation only under nonreducing conditions implying the key role of the disulfide bond in their amyloidogenicity. Such re-assembled amyloid is similar in terms of morphology and infrared features to typical bovine insulin fibrils, although it lacks the ability to seed the intact protein.