Spatiotemporal formation of a single liquid-like condensate and amyloid fibrils of α-synuclein by optical trapping at solution surface.

Liquid-like protein condensates have recently attracted much attention due to their critical roles in biological phenomena. They typically show high fluidity and reversibility for exhibiting biological functions, while occasionally serving as sites for the formation of amyloid fibrils. To comprehend the properties of protein condensates that underlie biological function and pathogenesis, it is crucial to study them at the single-condensate level; however, this is currently challenging due to a lack of applicable methods. Here, we demonstrate that optical trapping is capable of inducing the formation of a single liquid-like condensate of α-synuclein in a spatiotemporally controlled manner. The irradiation of tightly focused near-infrared laser at an air/solution interface formed a condensate under conditions coexisting with polyethylene glycol. The fluorescent dye-labeled imaging showed that the optically induced condensate has a gradient of protein concentration from the center to the edge, suggesting that it is fabricated through optical pumping-up of the α-synuclein clusters and the expansion along the interface. Furthermore, Raman spectroscopy and thioflavin T fluorescence analysis revealed that continuous laser irradiation induces structural transition of protein molecules inside the condensate to ß-sheet rich structure, ultimately leading to the condensate deformation and furthermore, the formation of amyloid fibrils. These observations indicate that optical trapping is a powerful technique for examining the microscopic mechanisms of condensate appearance and growth, and furthermore, subsequent aging leading to amyloid fibril formation.

Detection of Fibril Nucleation in Micrometer-Sized Protein Condensates and Suppression of Sup35NM Fibril Nucleation by Liquid-Liquid Phase Separation.

Elucidating the link between amyloid fibril formation and liquid-liquid phase separation (LLPS) is crucial in understanding the pathologies of various intractable human diseases. However, the effect of condensed protein droplets generated by LLPS on nucleation (the initial step of amyloid formation) remains unclear because of the lack of available quantitative analysis techniques. This study aimed to develop a measurement method for the amyloid droplet nucleation rate based on image analysis. We developed a method to fix micrometer-sized droplets in gel for long-term observation of protein droplets with known droplet volumes. By combining this method with image analysis, we determined the nucleation dynamics in droplets of a prion disease model protein, Sup35NM, at the single-event level. We found that the nucleation was unexpectedly suppressed by LLPS above the critical concentration (C*) and enhanced below C*. We also revealed that the lag time in the Thioflavin T assay, a semi-quantitative parameter of amyloid nucleation rate, does not necessarily reflect nucleation tendencies in droplets. Our results suggest that LLPS can suppress amyloid nucleation, contrary to the conventional hypothesis that LLPS enhances it. We believe that the proposed quantitative analytical method will provide insights into the role of LLPS from a pathological perspective.

Pathway Dependence of the Formation and Development of Prefibrillar Aggregates in Insulin B Chain.

Amyloid fibrils have been an important subject as they are involved in the development of many amyloidoses and neurodegenerative diseases. The formation of amyloid fibrils is typically initiated by nucleation, whereas its exact mechanisms are largely unknown. With this situation, we have previously identified prefibrillar aggregates in the formation of insulin B chain amyloid fibrils, which have provided an insight into the mechanisms of protein assembly involved in nucleation. Here, we have investigated the formation of insulin B chain amyloid fibrils under different pH conditions to better understand amyloid nucleation mediated by prefibrillar aggregates. The B chain showed strong propensity to form amyloid fibrils over a wide pH range, and prefibrillar aggregates were formed under all examined conditions. In particular, different structures of amyloid fibrils were found at pH 5.2 and pH 8.7, making it possible to compare different pathways. Detailed investigations at pH 5.2 in comparison with those at pH 8.7 have suggested that the evolution of protofibril-like aggregates is a common mechanism. In addition, different processes of evolution of the prefibrillar aggregates have also been identified, suggesting that the nucleation processes diversify depending on the polymorphism of amyloid fibrils.

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.

Current Understanding of the Structure, Stability and Dynamic Properties of Amyloid Fibrils.

Amyloid fibrils are supramolecular protein assemblies represented by a cross-ß structure and fibrous morphology, whose structural architecture has been previously investigated. While amyloid fibrils are basically a main-chain-dominated structure consisting of a backbone of hydrogen bonds, side-chain interactions also play an important role in determining their detailed structures and physicochemical properties. In amyloid fibrils comprising short peptide segments, a steric zipper where a pair of ß-sheets with side chains interdigitate tightly is found as a fundamental motif. In amyloid fibrils comprising longer polypeptides, each polypeptide chain folds into a planar structure composed of several ß-strands linked by turns or loops, and the steric zippers are formed locally to stabilize the structure. Multiple segments capable of forming steric zippers are contained within a single protein molecule in many cases, and polymorphism appears as a result of the diverse regions and counterparts of the steric zippers. Furthermore, the ß-solenoid structure, where the polypeptide chain folds in a solenoid shape with side chains packed inside, is recognized as another important amyloid motif. While side-chain interactions are primarily achieved by non-polar residues in disease-related amyloid fibrils, the participation of hydrophilic and charged residues is prominent in functional amyloids, which often leads to spatiotemporally controlled fibrillation, high reversibility, and the formation of labile amyloids with kinked backbone topology. Achieving precise control of the side-chain interactions within amyloid structures will open up a new horizon for designing useful amyloid-based nanomaterials.

Exploration of Insulin Amyloid Polymorphism Using Raman Spectroscopy and Imaging.

We aimed to investigate insulin amyloid fibril polymorphism caused by salt effects and heating temperature and to visualize the structural differences of the polymorphisms in situ using Raman imaging without labeling. The time course monitoring for amyloid formation was carried out in an acidic condition without any salts and with two species of salts (NaCl and Na2SO4) by heating at 60, 70, 80, and 90°C. The intensity ratio of two Raman bands at 1672 and 1657 cm-1 due to antiparallel ß-sheet and α-helix structures, respectively, was revealed to be an indicator of amyloid fibril formation, and the relative proportion of the ß-sheet structure was higher in the case with salts, especially at a higher temperature with Na2SO4. In conjunction with the secondary structural changes of proteins, the S-S stretching vibrational mode of a disulfide bond (∼514 cm-1) and the ratio of the tyrosine doublet I850/I826 were also found to be markers distinguishing polymorphisms of insulin amyloid fibrils by principal component analysis. Especially, amyloid fibrils with Na2SO4 media formed the gauche-gauche-gauche conformation of disulfide bond at a higher rate, but without any salts, the gauche-gauche-gauche conformation was partially transformed into the gauche-gauche-trans conformation at higher temperatures. The different environments of the hydroxyl groups of the tyrosine residue were assumed to be caused by fibril polymorphism. Raman imaging using these marker bands also successfully visualized the two- and three- dimensional structural differences of amyloid polymorphisms. These results demonstrate the potential of Raman imaging as a diagnostic tool for polymorphisms in tissues of amyloid-related diseases.

Structural Insights into the Inhibition of Amyloid Fibril Formation by Fibrinogen via Interaction with Prefibrillar Intermediates.

Abnormal protein aggregation tends to result in the formation of ß-sheet rich amyloid fibrils, which are related to various kinds of amyloidoses and neurodegenerative diseases. The susceptibility to aggregation of protein molecules is dealt with by proteostasis in living systems, in which molecular chaperones play an important role. Recently, several secreted proteins have been examined as extracellular chaperones with a potency to suppress the formation of amyloid fibrils, although the whole picture that includes their inhibition mechanisms is not yet understood. In this study, we investigated the inhibitory effect of fibrinogen (Fg), one of the extracellular proteins identified as a potential member of the group of chaperones, on fibril formation. Insulin B chain was used as an amyloid formation model system because its prefibrillar intermediate species in the nucleation phase were well characterized. We revealed that Fg efficiently inhibited amyloid fibril formation via a direct interaction with the surface of the prefibrillar intermediates. Small-angle X-ray scattering experiments and a stoichiometry analysis suggested a structural model in which the surface of the rod-shaped prefibrillar intermediates is surrounded by Fg molecules. From such a specific manner of interactions, we propose that the role of Fg is to disturb fibril growth by confining the nuclei even when the nucleation occurs inside the prefibrillar intermediate. The structural property of the B-chain intermediates complexed with Fg would provide insights into the general principles of the functions of chaperones and other potential chaperone-like proteins involved in amyloid-related diseases.

9-Aryl-3-aminocarbazole as an Environment- and Stimuli-Sensitive Fluorogen and Applications in Lipid Droplet Imaging.

Environment-sensitive luminophoric molecules have played an important role in the fields of smart materials, sensing, and bioimaging. In this study, it was demonstrated that depending on the substituents, 9-aryl-3-aminocarbazoles can display aggregation-induced emission and solvatofluorochromism, and the operating mechanism was clarified. The application of these compounds to lipid droplet imaging and fluorescent probes for cysteamine was demonstrated.

Heparin-dependent aggregation of hen egg white lysozyme reveals two distinct mechanisms of amyloid fibrillation.

Heparin, a biopolymer possessing high negative charge density, is known to accelerate amyloid fibrillation by various proteins. Using hen egg white lysozyme, we studied the effects of heparin on protein aggregation at low pH, raised temperature, and applied ultrasonic irradiation, conditions under which amyloid fibrillation was promoted. Heparin exhibited complex bimodal concentration-dependent effects, either accelerating or inhibiting fibrillation at pH 2.0 and 60 °C. At concentrations lower than 20 µg/ml, heparin accelerated fibrillation through transient formation of hetero-oligomeric aggregates. Between 0.1 and 10 mg/ml, heparin rapidly induced amorphous heteroaggregation with little to no accompanying fibril formation. Above 10 mg/ml, heparin again induced fibrillation after a long lag time preceded by oligomeric aggregate formation. Compared with studies performed using monovalent and divalent anions, the results suggest two distinct mechanisms of heparin-induced fibrillation. At low heparin concentrations, initial hen egg white lysozyme cluster formation and subsequent fibrillation is promoted by counter ion binding and screening of repulsive charges. At high heparin concentrations, fibrillation is caused by a combination of salting out and macromolecular crowding effects probably independent of protein net charge. Both fibrillation mechanisms compete against amorphous aggregation, producing a complex heparin concentration-dependent phase diagram. Moreover, the results suggest an active role for amorphous oligomeric aggregates in triggering fibrillation, whereby breakdown of supersaturation takes place through heterogeneous nucleation of amyloid on amorphous aggregates.

Femtosecond-Laser-Enhanced Amyloid Fibril Formation of Insulin.

Femtosecond (fs)-laser-induced crystallization as a novel crystallization technique was proposed for the first time by our group, where the crystallization time can be significantly shortened under fs laser irradiation. Similarly, we have further extended our investigation to amyloid fibril formation, also known as a nucleation-dependence process. Here we demonstrate that the necessary time for amyloid fibril formation can be significantly shortened by fs laser irradiation, leading to favorable enhancement. The enhancement was confirmed by both spectral measurements and direct observations of amyloid fibrils. The thioflavin T fluorescence intensity of laser-irradiated solution increased earlier than that of the control solution, and such a difference was simultaneously revealed by ellipticity changes. At the same time before intensity saturation in fluorescence, the number of amyloid fibrils obtained under laser irradiation was generally more than that in the control solution. Besides, such an enhancement is correlated to the laser power threshold of cavitation bubbling. Possible mechanisms are proposed by referring to fs-laser-induced crystallization and ultrasonication-induced amyloid fibril formation.

Stepwise organization of the ß-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-ß 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 ß-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 ß-sheet regions between the prefibrillar intermediate and mature fibrils using proteolysis led to the proposal of specific regions essential for manifestation of these properties.

Oligomerization and aggregation of NAP-22 with several metal ions.

Metal ions participate in various biochemical processes such as electron transport chain, gene transcription, and enzymatic reactions. Furthermore, the aggregation promoting effect of several metal ions on neuronal proteins such as prion, tau, Aß peptide, and α-synuclein, has been reported. NAP-22 (also called BASP1 or CAP-23) is a neuron-enriched calmodulin-binding protein and one of the major proteins in the detergent-resistant membrane microdomain fraction of the neuronal cell membrane. Previously, we showed oligomer formation of NAP-22 in the presence of several phospholipids and fatty acids. In this study, we found the aggregation of NAP-22 by FeCl2, FeCl3, and AlCl3 using native-PAGE. Oligomer or aggregate formation of NAP-22 by ZnCl2 or CuSO4 was shown with SDS-PAGE after cross-linking with glutaraldehyde. Morphological analysis with electron microscopy revealed the formation of large aggregates composed of small annular oligomers in the presence of FeCl3, AlCl3, or CuSO4. In case of FeCl2 or ZnCl2, instead of large aggregates, scattered annular and globular oligomers were observed. Interestingly, metal ion induced aggregation of NAP-22 was inhibited by several coenzymes such as NADP+, NADPH, or thiamine pyrophosphate. Since NAP-22 is highly expressed in the presynaptic region of the synapse, this result suggests the participation of metal ions not only on the protein and membrane dynamics at the presynaptic region, but also on the metabolic regulation though the interaction with coenzymes.

Fusion of amyloid beta with ferritin yields an isolated oligomeric beta-sheet-rich aggregate inside the ferritin cage.

Alzheimer's disease is a severe brain condition caused by the formation of amyloid plaques composed of amyloid beta (Aß) peptides. These peptides form oligomers, protofibrils, and fibrils before deposition into amyloid plaques. Among these intermediates, Aß oligomers (AßOs) were found to be the most toxic and therefore an appealing target for drug development and understanding their role in the disease. However, precise isolation and characterization of AßOs have proven challenging because AßOs tend to aggregate and form heterogeneous mixtures in solution. As a solution, we genetically fused the Aß peptide with a ferritin monomer. Such fusion allowed the encapsulation of precisely 24 Aß peptides inside the 24-mer ferritin cage. Using high-speed atomic force microscopy (HS-AFM), we disassembled ferritin and directly visualized the Aß core enclosed within the cage. The thioflavin-T assay (ThT) and attenuated total reflection infrared spectroscopy (ATR-IR) revealed the presence of a ß-sheet structure in the encapsulated oligomeric aggregate. Gallic acid, an amyloid inhibitor, can inhibit the fluorescence of ThT bound AßOs. Our approach represents a significant advancement in the isolation and characterization of ß-sheet rich AßOs and is expected to be useful for future studies of other disordered peptides such as α-synuclein and tau.

Mechanistic Modeling of Amyloid Oligomer and Protofibril Formation in Bovine Insulin.

Early phase of amyloid formation, where prefibrillar aggregates such as oligomers and protofibrils are often observed, is crucial for understanding pathogenesis. However, the detailed mechanisms of their formation have been difficult to elucidate because they tend to form transiently and heterogeneously. Here, we found that bovine insulin protofibril formation proceeds in a monodisperse manner, which allowed us to characterize the detailed early aggregation process by light scattering in combination with thioflavin T fluorescence and Fourier transform infrared spectroscopy. The protofibril formation was specific to bovine insulin, whereas no significant aggregation was observed in human insulin. The kinetic analysis combining static and dynamic light scattering data revealed that the protofibril formation process in bovine insulin can be divided into two steps based on fractal dimension. When modeling the experimental data based on Smoluchowski aggregation kinetics, an aggregation scheme consisting of initial fractal aggregation forming spherical oligomers and their subsequent end-to-end association forming protofibrils was clarified. Furthermore, the analysis of temperature and salt concentration dependencies showed that the end-to-end association is the rate-limiting step, involving dehydration. The established model for protofibril formation, wherein oligomers are incorporated as a precursor, provides insight into the molecular mechanism by which protein molecules assemble during the early stage of amyloid formation.

Polymorphism of ß2-microglobulin amyloid fibrils manifested by ultrasonication-enhanced fibril formation in trifluoroethanol.

The polymorphic property of amyloid structures has been focused on as a molecular basis of the presence and propagation of different phenotypes of amyloid diseases, although little is known about the molecular mechanism for expressing diverse structures from only one protein sequence. Here, we have found that, in combination with an enhancing effect of ultrasonication on nucleation, ß(2)-microglobulin, a protein responsible for dialysis-related amyloidosis, generates distinct fibril conformations in a concentration-dependent manner in the presence of 2,2,2-trifluoroethanol (TFE). Although the newly formed fibrils all exhibited a similar needle-like morphology with an extensive cross-ß core, as suggested by Fourier transform infrared absorption spectra, they differed in thioflavin T intensity, extension kinetics, and tryptophan fluorescence spectra even in the same solvents, representing polymorphic structures. The hydrophobic residues seemed to be more exposed in the fibrils originating at higher concentrations of TFE, as indicated by the increased binding of 1-anilinonaphthalene-8-sulfonic acid, suggesting that the modulation of hydrophobic interactions is critical to the production of polymorphic amyloid structures. Interestingly, the fibrils formed at higher TFE concentrations showed significantly higher stability against guanidium hydrochloride, the perturbation of ionic strength, and, furthermore, pressurization. The cross-ß structure inside the fibrils seems to have been more idealized, resulting in increased stability when nucleation occurred in the presence of the alcohol, indicating that a weaker contribution of hydrophobic interactions is intrinsically more amenable to the formation of a non-defective amyloid structure.

Hyper Thermostability and Liquid-Crystal-Like Properties of Designed α-Helical Peptide Nanofibers.

Structural and thermodynamic transitions of artificially designed α-helical nanofibers were investigated using eight peptide variants, including four peptides with amide-modified carboxyl termini (CB peptides) and four unmodified peptides (CF peptides). Temperature-dependent circular dichroism spectroscopy and differential scanning calorimetry showed that CB peptides exhibit thermostability up to 50 °C higher than CF peptides. As a result, one of the denaturation temperatures approached nearly 130 °C, which is exceptionally high for a biomacromolecule. Thermodynamic analysis and microscopy observations also showed that CB peptides undergo a thermal transition similar to the phase transition in liquid crystals. In addition, one of the peptides showed a sharp and highly cooperative transition with a small enthalpy change at around 25 °C, which was ascribed to a giga-bundle burst of the molecular assembly. These macroscopic changes in the thermostability and crystallinity of CB peptides may be attributed to an increased amphiphilicity of the molecule in the direction of the helix axis, originating from the microscopic modification of the carboxyl-terminus.

Variations in the Protein Hydration and Hydrogen-Bond Network of Water Molecules Induced by the Changes in the Secondary Structures of Proteins Studied through Near-Infrared Spectroscopy.

This study investigated how the secondary structural changes of proteins in aqueous solutions affect their hydration and the hydrogen-bond network of water molecules using near-infrared (NIR) spectroscopy. The aqueous solutions of three types of proteins, i.e., ovalbumin, ß-lactoglobulin, and bovine serum albumin, were denatured by heating, and changes in the NIR bands of water reflecting the states of hydrogen bonds induced via protein secondary structural changes were investigated. On heating, the intermolecular hydrogen bonds between water molecules as well as between water and protein molecules were broken, and protein molecules were no longer strongly bound by the surrounding water molecules. Consequently, the denaturation was observed to proceed depending on the thermodynamic properties of the proteins. When the aqueous solutions of proteins were cooled after denaturation, the hydrogen-bond network was reformed. However, the state of protein hydration was changed owing to the secondary structural changes of proteins, and the variation patterns were different depending on the protein species. These changes in protein hydration may be derived from the differences in the surface charges of proteins. The elucidation of the mechanism of protein hydration and the formation of the hydrogen-bond network of water molecules will afford a comprehensive understanding of the protein functioning and dysfunctioning derived from the structural changes in proteins.

Ultrasonication-dependent production and breakdown lead to minimum-sized amyloid fibrils.

Because of the insolubility and polymeric properties of amyloid fibrils, techniques used conventionally to analyze protein structure and dynamics have often been hampered. Ultrasonication can induce the monomeric solution of amyloidogenic proteins to form amyloid fibrils. However, ultrasonication can break down preformed fibrils into shorter fibrils. Here, combining these 2 opposing effects on beta(2)-microglobulin (beta2-m), a protein responsible for dialysis-related amyloidosis, we present that ultrasonication pulses are useful for preparing monodispersed amyloid fibrils of minimal size with an average molecular weight of approximately 1,660,000 (140-mer). The production of minimal and monodispersed fibrils is achieved by the free energy minimum under competition between fibril production and breakdown. The small homogeneous fibrils will be of use for characterizing the structure and dynamics of amyloid fibrils, advancing molecular understanding of amyloidosis.

Multistep growth of amyloid intermediates and its inhibition toward exploring therapeutic way: A case study using insulin B chain and fibrinogen.

It is crucial to understand the mechanism of amyloid fibril formation for the development of the therapeutic ways against amyloidoses and neurodegenerative diseases. Prefibrillar intermediates, which emerge prior to the fibril formation, seem to play a key role to the occurrence of nuclei of amyloid fibrils. We have focused on an insulin-derived peptide, B chain, to precisely clarify the mechanism of the fibril formation via prefibrillar intermediates. Various kinds of methods such as circular dichroism spectroscopy, dynamic light scattering, small-angle X-ray scattering, and atomic force microscopy were employed to track the structural changes in prefibrillar intermediates. The prefibrillar intermediates possessing rod-shaped structures elongated as a function of time, which led to fibril formation. We have also found that a blood clotting protein, fibrinogen, inhibits the amyloid fibril formation of B chain. This was caused by the stabilization of prefibrillar intermediates and thus the suppression of their elongation by fibrinogen. These findings have not only shed light on detailed mechanisms about how prefibrillar intermediates convert to the amyloid fibril, but also demonstrated that inhibiting the structural development of prefibrillar intermediates is an effective strategy to develop therapeutic ways against amyloid-related diseases. This review article is an extended version of the Japanese article, Observing Development of Amyloid Prefibrillar Intermediates and their Interaction with Chaperones for Inhibiting the Fibril Formation, published in SEIBUTSU BUTSURI Vol. 61, p. 236-239 (2021).

Tracking the Structural Development of Amyloid Precursors in the Insulin B Chain and the Inhibition Effect by Fibrinogen.

Amyloid fibrils are abnormal protein aggregates associated with several amyloidoses and neurodegenerative diseases. Prefibrillar intermediates, which emerge before amyloid fibril formation, play an important role in structure formation. Therefore, to prevent fibril formation, the mechanisms underpinning the structural development of prefibrillar intermediates must be elucidated. An insulin-derived peptide, the insulin B chain, is known for its stable accumulation of prefibrillar intermediates. In this study, the structural development of B chain prefibrillar intermediates and their inhibition by fibrinogen (Fg) were monitored by transmission electron microscopy (TEM) and small-angle X-ray scattering (SAXS) combined with solid-state nuclear magnetic resonance spectroscopy (NMR) and size exclusion chromatography. TEM images obtained in a time-lapse manner demonstrated that prefibrillar intermediates were wavy rod-like structures emerging from initial non-rod-like aggregates, and their bundling was responsible for protofilament formation. Time-resolved SAXS revealed that the prefibrillar intermediates became thicker and longer as a function of time. Solid-state NMR measurement suggested a ß-sheet formation around Ala14 residue was crucial for the structural conversion from prefibrillar intermediates to amyloid fibril. These observations suggested that prefibrillar intermediates serve as reaction fields for amyloid nucleation and its structural propagation. Time-resolved SAXS also demonstrated that Fg prevented elongation of the prefibrillar intermediates by forming specific complexes together, which implied that regulation of the length of prefibrillar intermediates upon Fg binding was the factor suppressing the prefibrillar intermediate elongation. The fibril formation mechanism and the inhibition strategy found in this study will be helpful in seeking appropriate methods against amyloid-related diseases.