Amyloid fibrils as functionalizable components of nanocomposite materials

Amyloid fibrils are a form of protein nanofiber that show promise as components of multifunctional bionanomaterials. In this work, native bovine insulin and bovine insulin that had been previously converted into amyloid fibrils were combined with poly(vinyl alcohol) (PVOH) via solution casting to determine the effect of fibrillization on the thermomechanical properties of the resulting composite. The synthesis method was found to preserve the amyloid fibril structure and properties of the resulting fibril-PVOH composite were investigated. At a filling level of 0.6 wt %, the fibril-reinforced PVOH was 15% stiffer than the PVOH control. Various properties of the films, including the glass transition temperature, degradation temperature, microstructure, and film morphology were characterized. Although more work is required to optimize the properties of the composites, this study provides proof of principle that incorporation of amyloid fibrils into a polymeric material can impart useful changes to the mechanical and morphological properties of the films.     

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.     

Controlling amyloid fibril formation by partial stirring

Many proteins undergoe self‐assembly into fibrillar structures known as amyloid fibrils. During the self‐assembly process, related structures known as spherulites can be formed. Herein we report a facile method where the balance between amyloid fibrils and spherulites can be controlled by stirring of the reaction mixture during the initial stages of the self‐assembly process. Moreover, we report how this methodology can be used to prepare non‐covalently functionalized amyloid fibrils. By stirring the reaction mixture continuously or for a limited time during the lag phase, the fibril length, and hence the propensity to form liquid crystalline phases, can be influenced. This phenomena is utilized in order to prepare films consisting of aligned protein fibrils incorporating the laser dye Nile red. The resulting films display polarized Nile red fluorescence. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 249–259, 2016.     

In vitro formation of amyloid fibrils from intact beta 2-microglobulin

Prompted by the identification of hemodialysis-associated amyloid protein as beta 2-microglobulin, we attempted to create in vitro amyloid fibrils from the native protein. Beta 2-microglobulin in PBS was slowly dialyzed free of salt and then concentrated. The residue showed Congophilia with green birefringence by light microscopy and polarization, and non-branching fibrils of indeterminate length, measuring 8 to 10 nm in diameter by electron microscopy, thus meeting the morphologic criteria for amyloid. The present study demonstrates the first successful in vitro creation of amyloid fibrils with intact precursor protein molecules and provides supporting evidence that hemodialysis-associated amyloid is constituted from beta 2-microglobulin.     

Characterization of thin layers of glucose oxidase

Glucose Oxidase (GOD) has been covalently bound to functionalized glass cover slips. The surface density of immobilized GOD molecules was measured by a method based on the amperometric determination of Flavin Adenine Dinucleotide (FAD). Atomic Force Microscopy (AFM) images, obtained in aqueous solution for the covalently bound enzyme, show a monomolecular layer of the enzyme on a functionalized glass surface. The catalytic constants were measured for the immobilized GOD and compared with those of the free enzyme.     

Coaggregation of amyloid fibrils for the preparation of stable and immobilized enzymes

Highly stable enzyme coaggregates were developed using amyloid fibrils as support materials. Amyloid fibril formation was induced by ionic liquids, and immobilization was done by the coaggregation of enzymes and amyloid fibrils followed by chemical cross-linking. Transmission and scanning electron microscopy studies were carried out to characterize the coaggregates. The amyloid fibril-linked enzymes showed significantly increased stability against various deactivating conditions. In addition, a high level of reusability was clearly observed. This study clearly demonstrated that amyloid fibrils can be used as biomaterials for enzyme immobilization and that amyloid fibril-linked enzyme coaggregates have good potential for industrial applications.     

Molecularly Ordered Bioelectrocatalytic Composite Inside a Film of Aligned Carbon Nanotubes

Molecularly ordered composites of polyvinylimidazole‐[Os(bipyridine)₂Cl] (PVI‐[Os(bpy)₂Cl]) and glucose oxidase (GOD) are assembled inside a film of aligned carbon nanotubes. The structure of the prepared GOD/PVI‐[Os(bpy)₂Cl]/CNT composite film is entirely uniform and stable; more than 90% bioelectrocatalytic activity could be maintained even after storage for 6 d. Owing to the ideal positional relationship achieved between enzyme, mediator, and electrode, the prepared film shows a high bioelectrocatalytic activity for glucose oxidation (ca. 15 mA cm^?2 at 25 °C) with an extremely high electron‐transfer turnover rate (ca. 650 s^?1) comparable to the value for GOD solutions, indicating almost every enzyme molecule entrapped within the ensemble (ca. 3 × 10¹² enzymes in a 1 mm × 1 mm film) can work to the fullest extent. This free‐standing, flexible composite film can be used by winding on a needle device; as an example, a self‐powered sugar monitor is demonstrated.     

Determination of amyloid core structure using chemical shifts

Amyloid fibrils are the pathological hallmark of a large variety of neurodegenerative disorders. The structural characterization of amyloid fibrils, however, is challenging due to their non‐crystalline, heterogeneous, and often dynamic nature. Thus, the structure of amyloid fibrils of many proteins is still unknown. We here show that the structure calculation program CS‐Rosetta can be used to obtain insight into the core structure of amyloid fibrils. Driven by experimental solid‐state NMR chemical shifts and taking into account the polymeric nature of fibrils CS‐Rosetta allows modeling of the core of amyloid fibrils. Application to the Y145X stop mutant of the human prion protein reveals a left‐handed β‐helix     

Detection of synergistic interactions of polyvinyl alcohol–cassava starch blends through DSC

The synergistic interaction of polyvinyl alcohol (PVOH) and cassava starch was studied by differential scanning calorimetry (DSC) method. Film of the PVOH–cassava starch blends were prepared by solution cast method. Originally, cassava starch film did not show presence of any endothermic peaks in DSC thermogram. However, after adding PVOH to cassava starch, the PVOH–cassava starch blend films showed obvious endothermic peaks with onset and end-point temperatures higher than neat PVOH film. In addition, the PVOH–cassava starch blends have experimental enthalpy of melting higher than theoretical values. This evidence shows that the interactions between PVOH and cassava starch molecules are extensively strong. Due to the synergistic interactions of PVOH and cassava starch, it is postulated that incorporation of 65–75 wt.% of PVOH in cassava starch blend has physical bonding equivalent to neat PVOH.     

Structure and biosensor characteristics of complex between glucose oxidase and plasma-polymerized nanothin film

The structure and biosensor characteristics of complex between glucose oxidase (GOD) and plasma-polymerized nanothin film (PPF), in which the thickness is several nanometers, were investigated by atomic force microscopy (AFM) and electrochemical measurement. The GOD molecules were densely adsorbed onto the PPF surface treated by nitrogen plasma and the individual GOD molecules were observed. Subsequently, the GOD densely packed array on the PPF surface was subsequently treated by plasma polymerization (overcoating). AFM image showed that the thicker film gave the smoother surface, indicating that the GOD adsorbed on the surface was embedded more deeply in PPF. The amperometric biosensor characteristics of the GOD-PPF complex on a platinum electrode showed the current increment due to the enzymatic reaction with glucose addition, indicating that enzyme activity was retained although the enzyme has been exposed to the plasma gas related to diffusion of the substrate. This means that under mild exposure to organic plasma, the enzyme does not become seriously dysfunctional. Amperometric biosensor characteristics were strongly affected by monomer and thickness of PPF overcoating related with the diffusion of the substrate (glucose). Considering that the film deposition was performed using microfabrication-compatible organic plasma, our new method for protein architecture has a great potential of enabling high throughput production of bioelectronic devices.     

Thioflavin T interaction with amyloid fibrils as an instrument for their studying

Benzthiazole dye thioflavin T (ThT) is widely used to study the formation and structure of amyloid fibrils. Nevertheless, till now there is no common opinion concerning molecular mechanisms of ThT binding to amyloid fibrils and the reasons of dramatic increase in its fluorescence quantum yield on incorporation into amyloid fibrils. Our data prove that ThT molecules incorporate in the amyloid fibrils in the monomeric form and there is no ground to suppose the formation of ThT dimers, eximers, or micells. It was shown that the increase in the quantum yield of ThT incorporated in amyloid fibrils was caused by restriction of benzthiazole and aminobenzene rings torsion fluctuations relative to each other. The use of equilibrium microdialysis allowed determining the absorption spectrum, the number of binding modes of ThT with insulin amyloid fibrils and for each mode determining the binding constants and the number of binding sites for each mode.     

The ultrastructure of infectious L-type bovine spongiform encephalopathy prions constrains molecular models

Bovine spongiform encephalopathy (BSE) is a prion disease of cattle that is caused by the misfolding of the cellular prion protein (PrP^C) into an infectious conformation (PrP^Sc). PrP^C is a predominantly α-helical membrane protein that misfolds into a β-sheet rich, infectious state, which has a high propensity to self-assemble into amyloid fibrils. Three strains of BSE prions can cause prion disease in cattle, including classical BSE (C-type) and two atypical strains, named L-type and H-type BSE. To date, there is no detailed information available about the structure of any of the infectious BSE prion strains. In this study, we purified L-type BSE prions from transgenic mouse brains and investigated their biochemical and ultrastructural characteristics using electron microscopy, image processing, and immunogold labeling techniques. By using phosphotungstate anions (PTA) to precipitate PrP^Sc combined with sucrose gradient centrifugation, a high yield of proteinase K-resistant BSE amyloid fibrils was obtained. A morphological examination using electron microscopy, two-dimensional class averages, and three-dimensional reconstructions revealed two structural classes of L-type BSE amyloid fibrils; fibrils that consisted of two protofilaments with a central gap and an average width of 22.5 nm and one-protofilament fibrils that were 10.6 nm wide. The one-protofilament fibrils were found to be more abundant compared to the thicker two-protofilament fibrils. Both fibrillar assemblies were successfully decorated with monoclonal antibodies against N- and C-terminal epitopes of PrP using immunogold-labeling techniques, confirming the presence of polypeptides that span residues 100–110 to 227–237. The fact that the one-protofilament fibrils contain both N- and C-terminal PrP epitopes constrains molecular models for the structure of the infectious conformer in favour of a compact four-rung β-solenoid fold.     

Visualization of polymorphism in apolipoprotein C-II amyloid fibrils

The misfolding and self-assembly of proteins into amyloid fibrils, which occur in several debilitating and age-related diseases, are affected by common components of amyloid deposits, notably lipids and lipid complexes. Previously, the effects of phospholipids on amyloid fibril formation by apolipoprotein (apo) C-II have been examined, where low concentrations of micellar phospholipids and lipid bilayers induce a new, straight rod-like morphology for apoC-II fibrils. This fibril appearance is distinct from the twisted-ribbon morphology observed when apoC-II fibrils are formed in the absence of lipids. We used total internal reflection fluorescence microscopy (TIRFM) to visualize the described polymorphism of apoC-II amyloid fibrils. The spontaneous assembly of apoC-II into either twisted-ribbon fibrils in the absence of lipids or into fibrils of straight rod-like morphology when lipids are present was captured by TIRFM. The latter was found to be better suited for visualization using TIRFM. The difference between seeding of apoC-II straight fibrils on microscopic quartz slide and in test tube suggested a role for the effects of incubation surface on fibril formation. Seed-dependent growth of apoC-II straight fibrils was probed further by using a dual-labelling construct, giving insights into the straight fibril growth pattern.     

Reciprocal Interactions between Membrane Bilayers and S. aureus PSMα3 Cross-α Amyloid Fibrils Account for Species-Specific Cytotoxicity

Phenol-soluble modulin α3 (PSMα3) is a functional amyloid secreted by the pathogenic bacterium Staphylococcus aureus. This 22-residue peptide serves as a key virulence determinant, toxic to human cells via the formation of unique cross-α amyloid-like fibrils. We demonstrate that bilayer vesicles accelerated PSMα3 fibril formation, and the fibrils, in turn, inserted deeply into bilayers mimicking mammalian cell membranes, accounting for PSMα3 cellular toxicity. Importantly, a mere amphipathic helical conformation was not a sufficient determinant for membrane-activity of PSMα3, pointing to the functional role of cross-α fibrils. In contrast to deep insertion of PSMα3 into mammalian membrane bilayers, the peptide only interacted with the surface of bilayers mimicking bacterial membranes, which might be related to its lack of antibacterial activity. Together, our data provide mechanistic insight into species-specific toxicity of a key bacterial amyloid virulence factor via reciprocal interactions with membranes, and open new perspectives into amyloid-related cytotoxicity mediated by helical fibril structures.     

Bi-enzyme functionlized hollow PtCo nanochains as labels for an electrochemical aptasensor

In this work, a new signal amplification strategy based on hollow PtCo nanochains (HPtCoNCs) functionalized by bi-enzyme-horseradish peroxidase mimicking DNAzyme (HRP-DNAzyme) and glucose oxidase (GOD), as well as ferrocene-labeled secondary thrombin aptamer (Fc-TBA 2), is developed to construct a highly sensitive electrochemical aptasensor. The HRP-DNAzyme contains a special G-quadruplex structure with an intercalated hemin. With the surface area enlarged by HPtCoNCs, the amount of immobilized Fc-TBA 2, hemin and GOD can be enhanced. Under the enzyme catalysis of GOD, d-glucose is rapidly oxidized into gluconic acid accompanying with the generation of H?O?, which is further electrocatalyzed by Pt nanoparticles and HPR-DNAzyme to improve the electrochemical signal of Fc. With several amplification factors mentioned above, a wide linear ranged from 0.001 to 30 nM is acquired with a relatively low detection limit of 0.39 pM for thrombin. The present work demonstrates that using HPtCoNCs as labels is a promising way to amplify the analysis signal and improve the sensitivity of aptasensors.     

Nano‐mechanical characterization of disassembling amyloid fibrils using the Peak Force QNM method

The comprehensive understanding of disassembly mechanism of amyloid fibrils requires nano‐scale characterization of the mechanical properties of amyloid fibrils during the disassembly process. In this work, gemini surfactant C₁₂C₆C₁₂Br₂ micelles were used as a probe to disassemble Aβ(1‐40) fibrils. The microstructure evolution and nano‐mechanical properties of Aβ(1‐40) fibrils during the disassembly process were systematically investigated by the Peak Force Quantitative Nano‐mechanical (PF‐QNM) technique. The results show an obvious decrease in Young's modulus of mature fibrils with high β‐sheet contents (2.4 ± 1.0 GPa) in comparison to the resulting peptide/surfactant complexes (1.1 ± 0.8 GPa) with loose surface structures. Interestingly, the Young's modulus of spherical peptide/surfactant complexes on the core was more than 3 GPa. This strategy can be used as a standard protocol to investigate the interaction mechanism between amyloid fibrils and small molecules, which may open up new possibilities to explore the mechanism of relevant human diseases.     

Alzheimer's Aβ42 and Aβ40 peptides form interlaced amyloid fibrils

Deposition of amyloid β (Aβ) in the brain is a pathological hallmark of Alzheimer's disease. There are two major isoforms of Aβ: the 42‐residue Aβ42 and the 40‐residue Aβ40. The only difference between Aβ42 and Aβ40 is that Aβ42 has two extra residues at the C‐terminus. The amyloid plaques in Alzheimer's brains consist of mostly Aβ42 and some plaques contain only Aβ42, even though Aβ40 concentration is several‐fold more than Aβ42. Using electron paramagnetic resonance, we studied the formation of amyloid fibrils using a mixture of Aβ42 and Aβ40 in vitro. We show that Aβ42 and Aβ40 form mixed fibrils in an interlaced manner, although Aβ40 is not as efficient as Aβ42 in terms of being incorporated into Aβ42 fibrils. Our results suggest that both Aβ42 and Aβ40 would be present in amyloid plaques if in vivo aggregation of Aβ were similar to the in vitro process. Therefore, there must be some mechanisms that lead to the preferential deposition of Aβ42 at the extracellular space. Identifying such mechanisms may open new avenues for therapeutic interventions to treat Alzheimer's disease.     

Amyloid insulin interaction with erythrocytes.

Erythrocyte membrane interactions with insulin fibrils (amyloid) have been investigated using centrifugation, fluorescence spectroscopy, light scattering, and flow cytometric techniques. The results indicate that insulin fibrils are having moderate affinity to erythrocyte membrane. However, analysis of the apparent dissociation constants of human erythrocyte membranes (leaky and resealed vesicles) with amyloid insulin reveal that the insulin binding is drastically reduced on attaining the fibrillar state compared with native insulin. To understand the role of insulin receptors on erythrocytes binding to amyloid, we have studied the interaction of biotinylated forms of denatured and amyloidic insulin with erythrocytes. FITC-streptavidin was used as a counter staining in flow cytometry measurements. We found that insulin fibrils bind 10 times more with erythrocyte membranes than with amylin and denatured insulin.     

A structural core within apolipoprotein C-II amyloid fibrils identified using hydrogen exchange and proteolysis

Plasma apolipoproteins show alpha-helical structure in the lipid-bound state and limited conformational stability in the absence of lipid. This structural instability of lipid-free apolipoproteins may account for the high propensity of apolipoproteins to aggregate and accumulate in disease-related amyloid deposits. Here, we explore the properties of amyloid fibrils formed by apolipoproteins using human apolipoprotein (apo) C-II as a model system. Hydrogen-deuterium exchange and NMR spectroscopy of apoC-II fibrils revealed core regions between residues 19-37 and 57-74 with reduced amide proton exchange rates compared to monomeric apoC-II. The C-terminal core region was also identified by partial proteolysis of apoC-II amyloid fibrils using endoproteinase GluC and proteinase K. Complete tryptic hydrolysis of apoC-II fibrils followed by centrifugation yielded a single peptide in the pellet fraction identified using mass spectrometry as apoC-II(56-76). Synthetic apoC-II(56-76) readily formed fibrils, albeit with a different morphology and thioflavinT fluorescence yield compared to full-length apoC-II. Studies with smaller peptides narrowed this fibril-forming core to a region within residues 60-70. We postulate that the ability of apoC-II(60-70) to independently form amyloid fibrils drives fibril formation by apoC-II. These specific amyloid-forming regions within apolipoproteins may underlie the propensity of apolipoproteins and their peptide derivatives to accumulate in amyloid deposits in vivo.     

Fibril formation from the amyloid-β peptide is governed by a dynamic equilibrium involving association and dissociation of the monomer

Here I review the molecular mechanisms by which water-soluble monomeric amyloid-β (Aβ) peptides are transformed into well-organized supramolecular complexes called amyloid fibrils. The mechanism of amyloid formation is considered theoretically on the basis of experimental results, and the structural and mechanistic similarities of amyloid fibrils to three-dimensional crystals are highlighted. A number of important results from the literature are described. These include the observation that a correct ratio of monomer association and dissociation rate constants is key for formation of well-organized amyloid fibrils. The dynamic nature of the amyloid-β structure is discussed, along with the possibly obligate requirement of the transient formation of a hairpin-like fold prior to its incorporation into amyloid fibrils. Many rounds of monomer association and dissociation events may be present during an apparently silent lag-period. Amongst these association/dissociation events, interaction between the C-terminal regions of the Aβ peptide seems to be more favored. Such association and dissociation events occurring in a “trial-and-error” fashion may be an important requirement for the formation of well-organized amyloid fibrils.