Supersaturation-limited Amyloid Fibrillation of Insulin Revealed by Ultrasonication

Amyloid fibrils form in supersaturated solutions via a nucleation and growth mechanism. We proposed that ultrasonication may be an effective agitation to trigger nucleation that would otherwise not occur under the persistent metastability of supersaturation. However, the roles of supersaturation and effects of ultrasonication have not been elucidated in detail except for limited cases. Insulin is an amyloidogenic protein that is useful for investigating the mechanisms underlying amyloid fibrillation with biological relevance. We studied the alcohol-induced amyloid fibrillation of insulin using various concentrations of 2,2,2-trifluoroethanol and 1,1,1,3,3,3-hexafluoro-2-propanol at pH 2.0 and 4.8. Ultrasonic irradiation effectively triggered fibrillation under conditions in which insulin retained persistent supersaturation. Structural analyses by circular dichroism, Fourier transform infrared spectroscopy, transmission electron microscopy, and atomic force microscopy revealed that the dominant structures of fibrils varied between parallel and antiparallel β-sheets depending on the solvent conditions. pH and alcohol concentration-dependent phase diagrams showed a marked difference before and after the ultrasonic treatment, which indicated that the persistent metastability of supersaturation determined the conformations of insulin. These results indicate the importance of an alternative view of amyloid fibrils as supersaturation-limited crystal-like aggregates formed above the solubility limit.     

Heparin‐induced amyloid fibrillation of β2‐microglobulin explained by solubility and a supersaturation‐dependent conformational phase diagram

Amyloid fibrils are fibrillar deposits of denatured proteins associated with amyloidosis and are formed by a nucleation and growth mechanism. We revisited an alternative and classical view of amyloid fibrillation: amyloid fibrils are crystal‐like precipitates of denatured proteins formed above solubility upon breaking supersaturation. Various additives accelerate and then inhibit amyloid fibrillation in a concentration‐dependent manner, suggesting that the combined effects of stabilizing and destabilizing forces affect fibrillation. Heparin, a glycosaminoglycan and anticoagulant, is an accelerator of fibrillation for various amyloidogenic proteins. By using β₂‐microglobulin, a protein responsible for dialysis‐related amyloidosis, we herein examined the effects of various concentrations of heparin on fibrillation at pH 2. In contrast to previous studies that focused on accelerating effects, higher concentrations of heparin inhibited fibrillation, and this was accompanied by amorphous aggregation. The two‐step effects of acceleration and inhibition were similar to those observed for various salts. The results indicate that the anion effects caused by sulfate groups are one of the dominant factors influencing heparin‐dependent fibrillation, although the exact structures of fibrils and amorphous aggregates might differ between those formed by simple salts and matrix‐forming heparin. We propose that a conformational phase diagram, accommodating crystal‐like amyloid fibrils and glass‐like amorphous aggregates, is important for understanding the effects of various additives.     

Supersaturation-limited and Unlimited Phase Transitions Compete to Produce the Pathway Complexity in Amyloid Fibrillation

Although amyloid fibrils and amorphous aggregates are two types of aggregates formed by denatured proteins, their relationship currently remains unclear. We used β₂-microglobulin (β2m), a protein responsible for dialysis-related amyloidosis, to clarify the mechanism by which proteins form either amyloid fibrils or amorphous aggregates. When ultrasonication was used to accelerate the spontaneous fibrillation of β2m at pH 2.0, the effects observed depended on ultrasonic power; although stronger ultrasonic power effectively accelerated fibrillation, excessively strong ultrasonic power decreased the amount of fibrils formed, as monitored by thioflavin T fluorescence. An analysis of the products formed indicated that excessively strong ultrasonic power generated fibrillar aggregates that retained β-structures but without high efficiency as seeds. On the other hand, when the spontaneous fibrillation of β2m was induced at higher concentrations of NaCl at pH 2.0 with stirring, amorphous aggregates became more dominant than amyloid fibrils. These apparent complexities in fibrillation were explained comprehensively by a competitive mechanism in which supersaturation-limited reactions competed with supersaturation-unlimited reactions. We link the kinetics of protein aggregation and a conformational phase diagram, in which supersaturation played important roles.     

Acceleration of the depolymerization of amyloid β fibrils by ultrasonication

Amyloid fibrils, rigid and filamentous aggregates associated with various diseases, are often difficult to depolymerize into monomers. Ultrasonication is a strong agitation that accelerates nucleation above the critical concentration of amyloid fibrillation. We examined the effects of ultrasonication on the fibrils of amyloid β(1–40) as well as on monomers. Ultrasonic pulses accelerated spontaneous fibrillation when the peptide concentration was above 1 μM. On the other hand, ultrasonic pulses accelerated the depolymerization of fibrils into monomers at 1 μM. These results indicate that, although amyloid fibrillation is a reversible process determined by thermodynamic stability, kinetically trapped supersaturation and physical difficulty of dissolving rigid fibrils prevent the smooth phase transitions. We propose that, in addition to accelerating the nucleation of fibrillation and fragmentation of fibrils above the critical concentration, ultrasonication is useful for dissolving fibrils below the critical concentration.     

Ultrasonication-dependent acceleration of amyloid fibril formation

Amyloid fibrils, similar to crystals, form through nucleation and growth. Because of the high free-energy barrier of nucleation, the spontaneous formation of amyloid fibrils occurs only after a long lag phase. Ultrasonication is useful for inducing amyloid nucleation and thus for forming fibrils, while the use of a microplate reader with thioflavin T fluorescence is suitable for detecting fibrils in many samples simultaneously. Combining the use of ultrasonication and microplate reader, we propose an efficient approach to studying the potential of proteins to form amyloid fibrils. With β₂-microglobulin, an amyloidogenic protein responsible for dialysis-related amyloidosis, fibrils formed within a few minutes at pH 2.5. Even under neutral pH conditions, fibrils formed after a lag time of 1.5 h. The results propose that fibril formation is a physical reaction that is largely limited by the high free-energy barrier, which can be effectively reduced by ultrasonication. This approach will be useful for developing a high-throughput assay of the amyloidogenicity of proteins.     

Elongation of amyloid fibrils through lateral binding of monomers revealed by total internal reflection fluorescence microscopy

Amyloid fibrils are fibrillar aggregates of denatured proteins associated with a large number of amyloidoses. The formation of amyloid fibrils has been considered to occur by nucleation and elongation. Real-time imaging of the elongation as well as linear morphology of amyloid fibrils suggests that all elongation events occur at the growing ends of fibrils. On the other hand, we suggested that monomers also bind to the lateral sides of preformed fibrils during the seed-dependent elongation, diffuse to the growing ends, and finally make further conformation changes to the mature amyloid fibrils. To examine lateral binding during the elongation of fibrils, we used islet amyloid polypeptide (IAPP), which has been associated with type II diabetes, and prepared IAPP modified with the fluorescence dye, Alexa532. By monitoring the elongation process with amyloid specific thioflavin T and Alexa532 fluorescence, we obtained overlapping images of the two fluorescence probes, which indicated lateral binding. These results are similar to the surface diffusion-dependent growth of crystals, further supporting the similarities between amyloid fibrillation and the crystallization of substances.     

A common mechanism underlying amyloid fibrillation and protein crystallization revealed by the effects of ultrasonication

Protein crystals form in supersaturated solutions via a nucleation and growth mechanism. The amyloid fibrils of denatured proteins also form via a nucleation and growth mechanism. This similarity suggests that, although protein crystals and amyloid fibrils are distinct in their morphologies, both processes can be controlled in a similar manner. It has been established that ultrasonication markedly accelerates the formation of amyloid fibrils and simultaneously breaks them down into fragmented fibrils. In this study, we investigated the effects of ultrasonication on the crystallization of hen egg white lysozyme and glucose isomerase from Streptomyces rubiginosus. Protein crystallization was monitored by light scattering, tryptophan fluorescence, and light transmittance. Repeated ultrasonic irradiations caused the crystallization of lysozyme and glucose isomerase after cycles of irradiations. The size of the ultrasonication-induced crystals was small and homogeneous, and their numbers were larger than those obtained under quiescent conditions. Switching off ultrasonic irradiation when light scattering or tryptophan fluorescence began to change resulted in the formation of larger crystals due to the suppression of the further nucleation and fractures in preformed crystals. The results indicate that protein crystallization and amyloid fibrillation are explained on the basis of a common phase diagram in which ultrasonication accelerates the formation of crystals or crystal-like amyloid fibrils as well as fragmentation of preformed crystals or fibrils.     

Polyphenol‐solubility alters amyloid fibril formation of α‐synuclein

Amyloid fibril formation is associated with various amyloidoses, including neurodegenerative diseases such as Alzheimer''s and Parkinson''s diseases. Amyloid fibrils form above the solubility of amyloidogenic proteins or peptides upon breaking supersaturation, followed by a nucleation and elongation mechanism, which is similar to the crystallization of solutes. Many additives, including salts, detergents, and natural compounds, promote or inhibit amyloid formation. However, the underlying mechanisms of the opposing effects are unclear. We examined the effects of two polyphenols, that is, epigallocatechin gallate (EGCG) and kaempferol‐7─O─glycoside (KG), with high and low solubilities, respectively, on the amyloid formation of α‐synuclein (αSN). EGCG and KG inhibited and promoted amyloid formation of αSN, respectively, when monitored by thioflavin T (ThT) fluorescence or transmission electron microscopy (TEM). Nuclear magnetic resonance (NMR) analysis revealed that, although interactions of αSN with soluble EGCG increased the solubility of αSN, thus inhibiting amyloid formation, interactions of αSN with insoluble KG reduced the solubility of αSN, thereby promoting amyloid formation. Our study suggests that opposing effects of polyphenols on amyloid formation of proteins and peptides can be interpreted based on the solubility of polyphenols.     

Small Liposomes Accelerate the Fibrillation of Amyloid β (1–40)

The deposition of amyloid β (Aβ) peptides is a pathological hallmark of Alzheimer disease. Aβ peptides were previously considered to interact specifically with ganglioside-containing membranes. Several studies have suggested that Aβ peptides also bind to phosphatidylcholine membranes, which lead to deformation of membranes and fibrillation of Aβ. Moreover, the role of membrane curvature, one type of deformation produced by binding of proteins to a membrane, in the binding and fibrillation of Aβ remains unclear. To clearly understand the relationship between the binding, consequent membrane deformation, and fibrillation of Aβ, we examined the amyloid fibrillation of Aβ-(1–40) in the presence of liposomes of various sizes. Membrane curvature increased with a decrease in the size of the liposomes. We used liposomes made of 1,2-dioleoyl-sn-glycero-3-phosphocholine to eliminate electrostatic effects. The results obtained showed that liposomes of smaller sizes (≤50 nm) significantly accelerated the nucleation step, thereby shortening the lag time of fibrillation. On the other hand, liposomes of larger sizes decreased the amount of fibrils but did not notably affect the lag time. The morphologies of fibrils, which were monitored by total internal reflection fluorescence microscopy, atomic force microscopy, and transmission electron microscopy, revealed that the length of Aβ-(1–40) fibrils became shorter and the amount of amorphous aggregates became larger as liposomes increased in size. These results suggest that the curvature of membranes coupled with an increase in water-accessible hydrophobic regions is important for binding and concentrating Aβ monomers, leading to amyloid nucleation. Furthermore, amyloid fibrillation on membranes may compete with non-productive binding to produce amorphous aggregates.     

Amyloid fibrillation kinetics: insight from atomistic nucleation theory

We consider the nucleation of nanosized amyloid fibrils composed of successively layered β-sheets at the molecular level when this process takes place by direct polymerization of protein segments (β-strands) into β-sheets. Application of the atomistic nucleation theory (ANT) to amyloid nucleation of β(2)-microglobulin and amyloid β(40) allows us to predict the fibril nucleus size and the fibril nucleation rate as functions of the supersaturation of the protein solution. The ANT predictions are compared to recent time-resolved optical experiments where they measure the effect of the protein concentration and mutations on the initial lag time before amyloid fibrils form in the protein solution. The presented analysis reveals the general principles underlying the nucleation kinetics of nanosized amyloid fibrils and indicates that it can be treated in the framework of existing general theories of the nucleation of new phases.     

BRICHOS Domains Efficiently Delay Fibrillation of Amyloid β-Peptide

Amyloid diseases such as Alzheimer, Parkinson, and prion diseases are associated with a specific form of protein misfolding and aggregation into oligomers and fibrils rich in β-sheet structure. The BRICHOS domain consisting of ～100 residues is found in membrane proteins associated with degenerative and proliferative disease, including lung fibrosis (surfactant protein C precursor; pro-SP-C) and familial dementia (Bri2). We find that recombinant BRICHOS domains from Bri2 and pro-SP-C prevent fibril formation of amyloid β-peptides (Aβ(40) and Aβ(42)) far below the stoichiometric ratio. Kinetic experiments show that a main effect of BRICHOS is to prolong the lag time in a concentration-dependent, quantitative, and reproducible manner. An ongoing aggregation process is retarded if BRICHOS is added at any time during the lag phase, but it is too late to interfere at the end of the process. Results from circular dichroism and NMR spectroscopy, as well as analytical size exclusion chromatography, imply that Aβ is maintained as an unstructured monomer during the extended lag phase in the presence of BRICHOS. Electron microscopy shows that although the process is delayed, typical amyloid fibrils are eventually formed also when BRICHOS is present. Structural BRICHOS models display a conserved array of tyrosine rings on a five-stranded β-sheet, with inter-hydroxyl distances suited for hydrogen-bonding peptides in an extended β-conformation. Our data imply that the inhibitory mechanism is reliant on BRICHOS interfering with molecular events during the lag phase.     

Dependence on solution conditions of aggregation and amyloid formation by an SH3 domain

The formation of amyloid fibrils by the SH3 domain of the alpha-subunit of bovine phosphatidylinositol-3'-kinase (PI3-SH3) has been investigated under carefully controlled solution conditions. NMR and CD characterisation of the denatured states from which fibrils form at low pH show that their properties can be correlated with the nature of the resulting aggregates defined by EM and FTIR spectroscopy. Compact partially folded states, favoured by the addition of anions, are prone to precipitate rapidly into amorphous species, whilst well-defined fibrillar structures are formed slowly from more expanded denatured states. Kinetic data obtained by a variety of techniques show a clear lag phase in the formation of amyloid fibrils. NMR spectroscopy shows no evidence for a significant population of small oligomers in solution during or after this lag phase. EM and FTIR indicate the presence of amorphous aggregates (protofibrils) rich in beta-structure after the lag phase but prior to the development of well-defined amyloid fibrils. These observations strongly suggest a nucleation and growth mechanism for the formation of the ordered aggregates. The morphologies of the fibrillar structures were found to be highly sensitive to the pH at which the protein solutions are incubated. This can be attributed to the effect of small perturbations in the electrostatic interactions that stabilise the contacts between the protofilaments forming the amyloid fibrils. Moreover, different hydrogen bonding patterns related to the various aggregate morphologies can be distinguished by FTIR analysis.     

Stepwise Organization of the β-Structure Identifies Key Regions Essential for the Propagation and Cytotoxicity of Insulin Amyloid Fibrils

Amyloid fibrils are supramolecular assemblies, the deposition of which is associated with many serious diseases including Alzheimer, prion, and Huntington diseases. Several smaller aggregates such as oligomers and protofibrils have been proposed to play a role in early stages of the fibrillation process; however, little is known about how these species contribute to the formation of mature amyloid fibrils with a rigid cross-β structure. Here, we identified a new pathway for the formation of insulin amyloid fibrils at a high concentration of salt in which mature fibrils were formed in a stepwise manner via a prefibrillar intermediate: minute prefibrillar species initially accumulated, followed by the subsequent formation of thicker amyloid fibrils. Fourier transform infrared spectra suggested the sequential formation of two types of β-sheets with different strength hydrogen bonds, one of which was developed concomitantly with the mutual assembly of the prefibrillar intermediate to form mature fibrils. Interestingly, fibril propagation and cellular toxicity appeared only after the later step of structural organization, and a comparison of β-sheet regions between the prefibrillar intermediate and mature fibrils using proteolysis led to the proposal of specific regions essential for manifestation of these properties.     

Nicking and fragmentation are responsible for α‐lactalbumin amyloid fibril formation at acidic pH and elevated temperature

Amyloid fibrils are ordered aggregates that may be formed from disordered, partially unfolded, and fragments of proteins and peptides. There are several diseases, which are due to the formation and deposition of insoluble β‐sheet protein aggregates in various tissue, collectively known as amyloidosis. Here, we have used bovine α‐lactalbumin as a model protein to understand the mechanism of amyloid fibril formation at pH 1.6 and 65°C under non‐reducing conditions. Amyloid fibril formation is confirmed by Thioflavin T fluorescence and atomic force microscopy (AFM). Our finding demonstrates that hydrolysis of peptide bonds occurs under these conditions, which results in nicking and fragmentation. The nicking and fragmentation have been confirmed on non‐reducing and reducing gel. We have identified the fragments by matrix‐assisted laser desorption ionization‐time of flight (MALDI‐TOF) mass spectrometry. The fragmentation may initiate nucleation as it coincides with AFM images. Conformational changes associated with monomer resulting in fibrillation are shown by circular dichroism and Raman spectroscopy. The current study highlights the importance of nicking and fragmentation in amyloid fibril formation, which may help understand the role of acidic pH and proteolysis under in vivo conditions in the initiation of amyloid fibril formation.     

Optical microscopy of growing insulin amyloid spherulites on surfaces in vitro

Amyloid fibrils are often found arranged into large ordered spheroid structures, known as spherulites, occurring in vivo and in vitro. The spherulites are predominantly composed of radially ordered amyloid fibrils, which self-assemble from protein in solution. We have observed and measured amyloid spherulites forming from heat-treated solutions of bovine insulin at low pH. The spherulites form in large numbers as semispherical dome-shaped objects on the cell surfaces, showing that surface defects or impurities, or the substrates themselves, can provide good nucleation sites for their formation. Using optical microscopy, we have measured the growth of individual spherulites as a function of time and in various conditions. There is a lag time before nucleation of the spherulites. Once they have nucleated, they grow, each with a radius increasing linearly, or faster than linearly, with time. Remarkably, this growth period has a sudden end, at which all spherulites in the system suddenly stop growing. A model of spherulite formation based on the polymerization of oriented fibrils around a nucleus, from a precursor in solution, quantitatively accounts for the observed growth kinetics. Seeding of native insulin solutions with preformed spherulites led to the preformed spherulites growing without a lag time. This seeding behavior is evidence that the fibrils in the spherulites assemble from small protein species rather than fibrils. The density of the spherulites was also measured and found to be constant with respect to radius, indicating that the space fills as the spherulite grows.     

Expanding the Repertoire of Amyloid Polymorphs by Co-polymerization of Related Protein Precursors

Amyloid fibrils can be generated from proteins with diverse sequences and folds. Although amyloid fibrils assembled in vitro commonly involve a single protein precursor, fibrils formed in vivo can contain more than one protein sequence. How fibril structure and stability differ in fibrils composed of single proteins (homopolymeric fibrils) from those generated by co-polymerization of more than one protein sequence (heteropolymeric fibrils) is poorly understood. Here we compare the structure and stability of homo and heteropolymeric fibrils formed from human β₂-microglobulin and its truncated variant ΔN6. We use an array of approaches (limited proteolysis, magic angle spinning NMR, Fourier transform infrared spectroscopy, and fluorescence) combined with measurements of thermodynamic stability to characterize the different fibril types. The results reveal fibrils with different structural properties, different side-chain packing, and strikingly different stabilities. These findings demonstrate how co-polymerization of related precursor sequences can expand the repertoire of structural and thermodynamic polymorphism in amyloid fibrils to an extent that is greater than that obtained by polymerization of a single precursor alone.     

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.     

Evidence for the existence of a secondary pathway for fibril growth during the aggregation of tau

The mechanism of amyloid fibril formation by proteins has been classically described by the nucleation-dependent polymerization (NDP) model, which makes certain predictions regarding the kinetics of fibrillation. All proteins whose aggregation conforms to the NDP model display a t(2) time dependence for their initial reaction profile. However, there are proteins whose aggregation reactions have kinetic signatures of a flat lag phase followed by an exponential rise in fibril mass, which does not conform to the NDP model. Amyloid fibril formation by tau, a microtubule-associated protein whose aggregation to form neurofibrillary tangles is implicated in Alzheimer's disease and other tauopathies, in the presence of inducers such as heparin and fatty acid micelles, has always been traditionally described by a ligand-induced NDP model. In this study, the existence of a secondary pathway for fibril growth during the aggregation of the functional, repeat domain of tau in the presence of heparin has been established. Both kinetic and accessory evidence are provided for the existence of this pathway, which is shown to augment the primary homogeneous nucleation pathway. From the kinetic data, the main secondary pathway that is operative appears to be fibril fragmentation but other pathways such as branching or secondary nucleation may also be operative.     

Amyloid nucleation triggered by agitation of beta2-microglobulin under acidic and neutral pH conditions

Amyloid nucleation through agitation was studied with beta2-microglobulin, which is responsible for dialysis-related amyloidosis, in the presence of salt under acid and neutral pH conditions. First, the aggregation of beta2-microglobulin in NaCl solutions was achieved by mildly agitating for 24 h at 37 degrees C protein solutions in three different states: acid-unfolded, salt-induced protofibrillar, and native. The formation of aggregates was confirmed by an increase in light scattering intensity of the solutions. Then, the aggregated samples were incubated without agitation at 37 degrees C for up to 25-45 days. The structural changes in the aggregated state during the incubation period were examined by means of fluorescence spectroscopy with thioflavin T, circular dichroism spectroscopy, and electron microscopy. The results revealed that all the samples in the different states produced a mature amyloid nucleus upon agitation, after which the fibrils elongated without any detectable lag phase during the incubation, with the acid-unfolded protein better suited to undergoing the structural rearrangements necessary to form amyloid fibrils than the more structured forms. The amount of aggregate including the amyloid nucleus produced by agitation from the native conformation at neutral pH was estimated to be about 9% of all the protein by an analysis using ultracentrifugation. Additionally, amyloid nucleation by agitation was similarly achieved for a different protein, hen egg-white lysozyme, in 0.5 M NaCl solution at neutral pH. Taken together, the agitation-treated aggregates of both proteins have a high propensity to produce an amyloid nucleus even at neutral pH, providing evidence that the aggregation pathway involves amyloid nucleation under entirely native conditions.     

Amyloid fibrils of human prion protein are spun and woven from morphologically disordered aggregates

Propagation and infectivity of prions in human prionopathies are likely associated with conversion of the mainly a-helical human prion protein, HuPrP, into an aggregated form with amyloid-like properties. Previous reports on efficient conversion of recombinant HuPrP have used mild to harsh denaturing conditions to generate amyloid fibrils in vitro. Herein we report on the in vitro conversion of four forms of truncated HuPrP (sequences 90–231 and 121–231 with and without an N-terminal hexa histidine tag) into amyloid-like fibrils within a few hours by using a protocol (phosphate buffered saline solutions at neutral pH with intense agitation) close to physiological conditions. The conversion process monitored by thioflavin T, ThT, revealed a three stage process with lag, growth and equilibrium phases. Seeding with preformed fibrils shortened the lag phase demonstrating the classic nucleated polymerization mechanism for the reaction. Interestingly, comparing thioflavin T kinetics with solubility and turbidity kinetics it was found that the protein initially formed non- thioflavionophilic, morphologically disordered aggregates that over time matured into amyloid fibrils. By transmission electron microscopy and by fluorescence microscopy of aggregates stained with luminescent conjugated polythiophenes (LCPs); we demonstrated that HuPrP undergoes a conformational conversion where spun and woven fibrils protruded from morphologically disordered aggregates. The initial aggregation functioned as a kinetic trap that decelerated nucleation into a fibrillation competent nucleus, but at the same time without aggregation there was no onset of amyloid fibril formation. The agitation, which was necessary for fibril formation to be induced, transiently exposes the protein to the air-water interface suggests a hitherto largely unexplored denaturing environment for prion conversion.Key words: misfolding, aggregation, amyloid, prion, conformational conversion, fluorescence