Mechanism Of Fibril Formation Research Articles

New clues into the self-assembly of Vmh2, a basidiomycota class I hydrophobin.

Hydrophobins are fungal proteins that can self-assemble into amphiphilic films at hydrophobic-hydrophilic interfaces. Class I hydrophobin aggregates resemble amyloid fibrils, sharing some features with them. Here, five site-directed mutants of Vmh2, a member of basidiomycota class I hydrophobins, were designed and characterized to elucidate the molecular determinants playing a key role in class I hydrophobin self-assembly. The mechanism of fibril formation proposed for Vmh2 foresees that the triggering event is the destabilization of a specific loop (L1), leading to the formation of a β-hairpin, which in turn generates the β-spine of the amyloid fibril.

Atomic structures of FUS LC domain segments reveal bases for reversible amyloid fibril formation.

Thermostable cross-β structures are characteristic of pathological amyloid fibrils, but these structures cannot explain the reversible nature of fibrils formed by RNA-binding proteins such as fused in sarcoma (FUS), involved in RNA granule assembly. Here, we find that two tandem (S/G)Y(S/G) motifs of the human FUS low-complexity domain (FUS LC) form reversible fibrils in a temperature- and phosphorylation-dependent manner. We named these motifs reversible amyloid cores, or RAC1 and RAC2, and determined their atomic structures in fibrillar forms, using microelectron and X-ray diffraction techniques. The RAC1 structure features an ordered-coil fibril spine rather than the extended β-strand typical of amyloids. Ser42, a phosphorylation site of FUS, is critical in the maintenance of the ordered-coil structure, which explains how phosphorylation controls fibril formation. The RAC2 structure shows a labile fibril spine with a wet interface. These structures illuminate the mechanism of reversible fibril formation and dynamic assembly of RNA granules.

Self-assembly of Mutant Huntingtin Exon-1 Fragments into Large Complex Fibrillar Structures Involves Nucleated Branching

Huntingtin (HTT) fragments with extended polyglutamine tracts self-assemble into amyloid-like fibrillar aggregates. Elucidating the fibril formation mechanism is critical for understanding Huntington's disease pathology and for developing novel therapeutic strategies. Here, we performed systematic experimental and theoretical studies to examine the self-assembly of an aggregation-prone N-terminal HTT exon-1 fragment with 49 glutamines (Ex1Q49). Using high-resolution imaging techniques such as electron microscopy and atomic force microscopy, we show that Ex1Q49 fragments in cell-free assays spontaneously convert into large, highly complex bundles of amyloid fibrils with multiple ends and fibril branching points. Furthermore, we present experimental evidence that two nucleation mechanisms control spontaneous Ex1Q49 fibrillogenesis: (1) a relatively slow primary fibril-independent nucleation process, which involves the spontaneous formation of aggregation-competent fibrillary structures, and (2) a fast secondary fibril-dependent nucleation process, which involves nucleated branching and promotes the rapid assembly of highly complex fibril bundles with multiple ends. The proposed aggregation mechanism is supported by studies with the small molecule O4, which perturbs early events in the aggregation cascade and delays Ex1Q49 fibril assembly, comprehensive mathematical and computational modeling studies, and seeding experiments with small, preformed fibrillar Ex1Q49 aggregates that promote the assembly of amyloid fibrils. Together, our results suggest that nucleated branching in vitro plays a critical role in the formation of complex fibrillar HTT exon-1 aggregates with multiple ends.

Revealing the Mechanism of Amyloid Fibril Formation by Combined Single Molecule FRET and Kinetic Modeling

The conversion of native soluble proteins into insoluble amyloid fibrils that are rich in cross β-sheet structure is associated with a variety of neurodegenerative diseases. It has recently become apparent that smaller oligomers formed during the early stages of amyloid fibril formation may be primarily responsible for the toxicity in amyloid diseases. Traditional ensemble techniques have limitations in characterization of oligomers due to the metastable and heterogeneous nature of these prefibrillar species. Single molecule fluorescence spectroscopy allows the conformational dynamics of individual biomolecules to be investigated, revealing properties that may be averaged in the ensemble experiment. It is therefore extremely powerful for identification and characterization of the low-populated, heterogeneous and transient species formed during fibril assembly. Combining single molecule fluorescence with kinetic modeling can provide quantitative information regarding the microscopic steps in amyloid formation. Here we have applied single molecule fluorescence resonance energy transfer to investigate in detail the intermolecular assembly and aggregation process of the yeast prion protein Ure2. Oligomerization during the initial lag phase was observed, and two types of Ure2 oligomers with different assembly modes were identified. Furthermore, using theoretical analysis combined with single molecule and ensemble kinetic data, we describe the formation and depletion pathway of oligomers, and propose a multi-step mechanism for Ure2 fibril formation, in which initial oligomerization is followed by conformational conversion to β-sheet containing oligomers that are then able to grow to form mature amyloid fibrils. We also show directly that secondary nuclei do not form, and that fragmentation is therefore responsible for the autocatalytic behavior of Ure2, in contrast to the mechanism of the amyloid formation of Aβ peptide. These results shed light on the potential origin of differences in cytotoxicity between functional and disease-related amyloids.

Structural mechanisms of oligomer and amyloid fibril formation by the prion protein.

Misfolding and aggregation of the prion protein is responsible for multiple neurodegenerative diseases. Works from several laboratories on folding of both the WT and multiple pathogenic mutant variants of the prion protein have identified several structurally dissimilar intermediates, which might be potential precursors to misfolding and aggregation. The misfolded aggregates themselves are morphologically distinct, critically dependent on the solution conditions under which they are prepared, but always β-sheet rich. Despite the lack of an atomic resolution structure of the infectious pathogenic agent in prion diseases, several low resolution models have identified the β-sheet rich core of the aggregates formed in vitro, to lie in the α2-α3 subdomain of the prion protein, albeit with local stabilities that vary with the type of aggregate. This feature article describes recent advances in the investigation of in vitro prion protein aggregation using multiple spectroscopic probes, with particular focus on (1) identifying aggregation-prone conformations of the monomeric protein, (2) conditions which trigger misfolding and oligomerization, (3) the mechanism of misfolding and aggregation, and (4) the structure of the misfolded intermediates and final aggregates.

NFGAIL Amyloid Oligomers: The Onset of Beta-Sheet Formation and the Mechanism for Fibril Formation.

The hexapeptide NFGAIL is a highly amyloidogenic peptide, derived from the human islet amyloid polypeptide (hIAPP). Recent investigations indicate that presumably soluble hIAPP oligomers are one of the cytotoxic species in type II diabetes. Here we use thioflavin T staining, transmission electron microscopy, as well as ion mobility-mass spectrometry coupled to infrared (IR) spectroscopy to study the amyloid formation mechanism and the quaternary and secondary structure of soluble NFGAIL oligomers. Our data reveal that at neutral pH NFGAIL follows a nucleation dependent mechanism to form amyloid fibrils. During the lag phase, highly polydisperse, polymorph, and compact oligomers (oligomer number n = 2-13) as well as extended intermediates (n = 4-11) are present. IR secondary structural analysis reveals that compact conformations adopt turn-like structures, whereas extended oligomers exhibit a significant amount of β-sheet content. This agrees well with previous molecular dynamic simulations and provides direct experimental evidence that unordered off-pathway NFGAIL aggregates up to the size of at least the 13-mer as well as partially folded β-sheet containing oligomers are coexisting.

Real-Time Observation of the Interaction between Thioflavin T and an Amyloid Protein by Using High-Sensitivity Rheo-NMR.

Amyloid fibril formation is associated with numerous neurodegenerative diseases. To elucidate the mechanism of fibril formation, the thioflavin T (ThT) fluorescence assay is widely used. ThT is a fluorescent dye that selectively binds to amyloid fibrils and exhibits fluorescence enhancement, which enables quantitative analysis of the fibril formation process. However, the detailed binding mechanism has remained unclear. Here we acquire real-time profiles of fibril formation of superoxide dismutase 1 (SOD1) using high-sensitivity Rheo-NMR spectroscopy and detect weak and strong interactions between ThT and SOD1 fibrils in a time-dependent manner. Real-time information on the interaction between ThT and fibrils will contribute to the understanding of the binding mechanism of ThT to fibrils. In addition, our method provides an alternative way to analyze fibril formation.

Unveiling the stimulatory effects of tartrazine on human and bovine serum albumin fibrillogenesis: Spectroscopic and microscopic study

Amyloid fibrils are playing key role in the pathogenesis of various neurodegenerative diseases. Generally anionic molecules are known to induce amyloid fibril in several proteins. In this work, we have studied the effect of anionic food additive dye i.e., tartrazine (TZ) on the amyloid fibril formation of human serum albumins (HSA) and bovine serum albumin (BSA) at pHs7.4 and 3.5. We have employed various biophysical methods like, turbidity measurements, Rayleigh Light Scattering (RLS), Dynamic Light Scattering (DLS), intrinsic fluorescence, Congo red assay, far-UV CD, transmission electron microscopy (TEM) and atomic force microscopy (AFM) to decipher the mechanism of TZ-induce amyloid fibril formation in both the serum albumins at pHs7.4 and 3.5. The obtained results suggest that both the albumins forms amyloid-like aggregates in the presence of 1.0 to 15.0mM of TZ at pH3.5, but no amyloid fibril were seen at pH7.4. The possible cause of TZ-induced amyloid fibril formation is electrostatic and hydrophobic interaction because sulfate group of TZ may have interacted electrostatically with positively charged amino acids of the albumins at pH3.5 and increased protein-protein and protein-TZ interactions leading to amyloid fibril formation. The TEM, RLS and DLS results are suggesting that BSA forms bigger size amyloids compared to HSA, may be due to high surface hydrophobicity of BSA.

Electric-Field-Oriented Growth of Long Hair-Like Silica Microfibrils and Derived Functional Monolithic Solids.

Controlled self-assembly using molecules/nanoparticles as building block materials represents an important approach for nanofabrication. We present a "bottom-up" fabrication approach to first grow a new class of inorganic (silica) long hair-like microfibrils or microwires and then to form monolithic solid pellet that contains parallel arrays of bundled microfibrils with a controlled orientation. During the sol-gel solution processing, reactive precursor species are utilized as molecular "building blocks" for the field-directed assembly growth of microfibrils driven by an electric field of pulsed direct current (dc) with controlled frequency. We have demonstrated a novel reactive electrofibrilation process that combines an external field with a solid-phase nucleation and growth process which in principle has no limitation on the type of reactions (such as the one here that involves sol-gel reaction chemistry) and on materials compositions (such as the example silica oxide), thus will enable bulk production of long microfibrils of wide variety of inorganic materials (other oxides or metals). Furthermore, we have fabricated uniquely architectured monolithic solid materials containing aligned microfibrils by "wet press" of the in-situ grown microfibril structure in the electric field. The consolidated monolithic slabs (1 cm × 1 cm × 3 mm) have shown anisotropic properties and desirable retention of DNA molecule fragments, thus, could serve as a platform stationary-phase materials for future development of capillary electrochromatography for biomolecule separations. Electrical field-guided self-assembly is an effective approach in producing long (hair-like) ceramic microfibrils, which can be further used in consolidation fabrication of oriented structured ceramic monoliths with potential for capillary electrophoretic chromatography and other separations applications. This original work was recorded through a patent application to understand the fibril formation mechanism and its process.

Rosetta Stone for Amyloid Fibrils: The Key Role of Ring-Like Oligomers in Amyloidogenesis

Deeper understanding of processes of protein misfolding, aggregation, formation of oligomers, protofibrils, and fibrils is crucial for the development of future medicine in treatment of amyloid-related diseases. While numerous reports illuminate the field, the above processes are extremely complex, as they depend on many varying parameters, such as the peptide concentration, temperature, pH, presence of metal ions, lipids, and organic solvents. Different mechanisms of amyloid fibril formation have been proposed, but the process of the oligomer-to-fibril transition is the least agreed upon. Our studies of a number of amyloidogenic proteins and peptides (insulin, Aβ peptides, the Bgl2 protein from the yeast cell wall), as well as their amyloidogenic fragments, have allowed us to propose a model of the fibril structure generation. We have found that the main building block of fibrils of any morphology is a ring-like oligomer. The varying models of interaction of ring oligomers with each other revealed in our studies make it possible to explain their polymorphism. Crucially, the amino acid sequence determines the oligomer structure for the given protein/peptide.

Cellular mechanism of fibril formation from serum amyloid A1 protein.

Serum amyloid A1 (SAA1) is an apolipoprotein that binds to the high-density lipoprotein (HDL) fraction of the serum and constitutes the fibril precursor protein in systemic AA amyloidosis. We here show that HDL binding blocks fibril formation from soluble SAA1 protein, whereas internalization into mononuclear phagocytes leads to the formation of amyloid. SAA1 aggregation in the cell model disturbs the integrity of vesicular membranes and leads to lysosomal leakage and apoptotic death. The formed amyloid becomes deposited outside the cell where it can seed the fibrillation of extracellular SAA1. Our data imply that cells are transiently required in the amyloidogenic cascade and promote the initial nucleation of the deposits. This mechanism reconciles previous evidence for the extracellular location of deposits and amyloid precursor protein with observations the cells are crucial for the formation of amyloid.

Biophysical evaluation of amyloid fibril formation in bovine cytochrome c by sodium lauroyl sarcosinate (sarkosyl) in acidic conditions

Surfactant-induced amyloid fibril formation in protein has a lot of association in the laboratory and industrial application. Sodium lauroyl sarcosinate (sarkosyl) is used to solubilize protein aggregates during protein purification, however, its amyloid fibril induction properties are very less understood. The aim of the current work is to examine the role of sarkosyl on the amyloid fibrillation of bovine cytochrome c protein at pH3.5. We have used various spectroscopic (turbidity, RLS, ThT and far-UV CD) and microscopic (TEM) techniques to characterize the fibrillation of cytochrome c at low pH. We have demonstrated that sarkosyl in the concentrations range 0.5 to 10.0mM is inducing large size aggregates in cytochrome c with fibrillar morphology. At concentrations of ≤0.5mM, sarkosyl was not able to induce amyloid-like aggregates in cytochrome c at the same pH. The α-helical content of cytochrome c is transformed into β-sheet structure (single minima at 218nm) in the presence of ≥0.5mM of sarkosyl. The amyloid fibril formation potency is dependent on the concentrations of sarkosyl and solution pH. We believe the results of this work will not only help to explain the molecular mechanism of amyloid fibril formation, but also give us an insight on rationally designing potential anti-amyloidogenic molecules.

Inhibition of Human Serum Albumin Fibrillation by Two-Dimensional Nanoparticles.

The formation and deposition of amyloid fibrils have been linked to the pathogenesis of numerous debilitating neurodegenerative disorders. Serum albumins serve as good model proteins for understanding the molecular mechanisms of protein aggregation and fibril formation. Graphene-based nanotherapeutics appear to be promising candidates for designing inhibitors of protein fibrillation. The inhibitory effect of graphene oxide (GO) nanoparticles on the fibrillation of human serum albumin (HSA) in an in vitro mixed solvent system has been investigated. The methods used include ThT fluorescence, ANS binding, Trp fluorescence, circular dichroism, fluorescence microscopy, field-emission scanning electron microscopy, and high-resolution transmission electron microscopy. It was observed that GO inhibits HSA fibrillation and forms agglomerates with β-sheet rich prefibrillar species. Binding of GO prevents the formation of mature fibrils with characteristic cross-β sheet but does not promote refolding to the native state.

Cationic gemini surfactant (16-4-16) interact electrostatically with anionic plant lectin and facilitates amyloid fibril formation at neutral pH

Amyloid are fibrous clumps or aggregates of protein which usually deposited in various organs and tissues which direct their degenration. In neurodenrative disease, these proteincious aggregates leads degeneration of neuronal tissues/organs. In order to develop drug condidate which can dissolve the amyloid fibrils and turned protein functional, it is urgent need to elucidate the mechanism of amyloid fibril formation under different conditions. In this study, we have taken a step to find the mechanism of amyloid fibril formation in concanavalin A (Con A) protein via cationic gemini surfactant (16-4-16) at two different pHs (7.4 and 3.5). We used several biophysical techniques such as Rayleigh light scattering, turbidity, ThT dye binding, intrinsic fluorescence, extrinsic fluorescence, far-UV CD and transmission electron microscopy to characterize the amyloid fibril formation of Con A by cationic gemini surfactant. The results suggest that the Con A form amyloid-like aggregates in the presence of very low gemini concentrations (2.5–125μM) at pH 7.4 while in the presence of higher concentrations (125–1000μM), Con A remained soluble. The Con A was not forming any aggregates or amyloid in the presence of same gemini concentrations at pH 3.5. The possible cause of gemini surfactant-induced amyloid fibril formation of Con A is electrostatic as well as hydrophobic interaction at pH 7.4 and strong electrostatic repulsion at pH 3.5. The far-UV CD spectra of Con A transformed into a cross β-sheet structure when incubated with low gemini surfactants while at higher concentrations the β-sheet structures of Con A transformed into α-helix.

Investigating the inhibitory effects of zinc ions on amyloid fibril formation of hen egg-white lysozyme

The amyloid fibrils derived from protein and peptide self-assembly have been studied in many diseases. In the present study, in combination with Thioflavin T(ThT) assay, Congo red(CR),transmission electron microscopy and cell cytotoxicity assay, we investigated the influence of zinc ions on amyloid fibril formation using hen egg white lysozyme (HEWL) as a model protein under high temperature and acidic pH conditions. We observed that HEWL tended to form the amyloid fibrils at pH 2.0 and 60°C, which was consistent with the previous studies. However, as the concentrations of zinc ions increased, the amounts of amyloid fibrils of HWEL gradually reduced, but the overall morphology of individual amyloid fibril was not significantly altered whether or not zinc ions were present. Moreover, by using circular dichroism (CD), ANS and intrinsic fluorescence spectra, we illustrated that zinc ions inhibited the formation of β-sheet and exposure of hydrophobic regions of HEWL. This work would help to understand the molecular mechanism of amyloid fibril formation.

Dynamic Aspects of Amyloid Fibrils of α-Synuclein Related to the Pathogenesis of Parkinson’s Disease

α-synuclein (αSyn) is a 140 amino acid protein of unknown function, abundant in presynaptic terminals of nerve cells. Filamentous aggregates (amyloid fibrils) of αSyn have been shown to be involved with the pathogenesis of Parkinson’s disease, a progressive neurodegenerative disorder. Elucidation of the mechanism of amyloid fibril formation of αSyn is thus important for elucidation of the pathogenesis mechanism of this disease. Amyloid fibril formation is observed for many proteins including, for example, the amyloid-β peptide, the prion protein, and transthyretin. Extensive studies on amyloid fibril formation have characterized structural and kinetic properties of these proteins during fibril formation. Whereas involvement of unfolding/misfolding of the proteins with fibril formation implies that the dynamics of the proteins plays an important role in fibril formation, the dynamic aspects of fibril formation have not been explored very much. In this review, dynamic behavior of αSyn in the monomeric and fibril states is described, based on our recent study on the dynamics of αSyn using quasielastic neutron scattering, by which the dynamics of proteins can be directly measured. It was found that diffusive global motions of the entire molecules and segmental motions within the molecules are observed in the monomeric state but largely suppressed in the fibril state. On the other hand, the amplitudes of the local motions such as side chain motions were found to be larger in the fibril state than in the monomeric state. This implies that significant solvent space exists within the fibrils, which is attributed to αSyn molecule within the fibrils having a distribution of conformations. The larger amplitudes of the side chain motions in the fibril state than in the monomeric state imply that the fibril state is entropically favorable. Implications of this unusual dynamic behavior of αSyn fibrils are discussed in terms of possible clinical relevance.

A pH-Induced Switch in Human Glucagon-like Peptide-1 Aggregation Kinetics.

Aggregation and amyloid fibril formation of peptides and proteins is a widespread phenomenon. It has serious implications in a range of areas from biotechnological and pharmaceutical applications to medical disorders. The aim of this study was to develop a better understanding of the mechanism of aggregation and amyloid fibrillation of an important pharmaceutical, human glucagon-like peptide-1 (GLP-1). GLP-1 is a 31-residue hormone peptide that plays an important role regulating blood glucose levels, analogues of which are used for treatment of type 2 diabetes. Amyloid fibril formation of GLP-1 was monitored using thioflavin T fluorescence as a function of peptide concentration between pH 7.5 and 8.2. Results from these studies establish that there is a highly unusual pH-induced switch in GLP-1 aggregation kinetics. At pH 8.2, the kinetics are consistent with a nucleation-polymerization mechanism for fibril formation. However, at pH 7.5, highly unusual kinetics are observed, where the lag time increases with increasing peptide concentration. We attribute this result to the formation of off-pathway species together with an initial slow, unimolecular step where monomer converts to a different monomeric form that forms on-pathway oligomers and ultimately fibrils. Estimation of the pKa values of all the ionizable groups in GLP-1 suggest it is the protonation/deprotonation of the N-terminus that is responsible for the switch with pH. In addition, a range of biophysical techniques were used to characterize (1) the start point of the aggregation reaction and (2) the structure and stability of the fibrils formed. These results show that the off-pathway species form under conditions where GLP-1 is most prone to form oligomers.

Elucidating Important Sites and the Mechanism for Amyloid Fibril Formation by Coarse-Grained Molecular Dynamics.

Fibrils formed by the β-amyloid (Aβ) peptide play a central role in the development of Alzheimer's disease. In this study, the principles governing their growth and stability are investigated by analyzing canonical and replica-exchange molecular dynamics trajectories of Aβ(9-40) fibrils. In particular, an unstructured monomer was allowed to interact freely with an Aβ fibril template. Trajectories were generated with the coarse-grained united-residue force field, and one- and two-dimensional free-energy landscapes (FELs) along the backbone virtual-bond angle θ and backbone virtual-bond-dihedral angle γ of each residue and principal components, respectively, were analyzed. Also, thermal unbinding (unfolding) of an Aβ peptide from the fibril template was investigated. These analyses enable us to illustrate the entire process of Aβ fibril elongation and to elucidate the key residues involved in it. Several different pathways were identified during the search for the fibril conformation by the monomer, which finally follows a dock-lock mechanism with two distinct locking stages. However, it was found that the correct binding, with native hydrogen bonds, of the free monomer to the fibril template at both stages is crucial for fibril elongation. In other words, if the monomer is incorrectly bound (with nonnative hydrogen bonds) to the fibril template during the first "docking" stage, it can remain attached to it for a long time before it dissociates and either attempts a different binding or allows another monomer to bind. This finding is consistent with an experimentally observed "stop-and-go" mechanism of fibril growth.

Common structural features of toxic intermediates from α-synuclein and GroES fibrillogenesis detected using cryogenic coherent X-ray diffraction imaging.

The aggregation and deposition of α-synuclein (αSyn) in neuronal cells is correlated to pathogenesis of Parkinson's disease. Although the mechanism of αSyn aggregation and fibril formation has been studied extensively, the structural hallmarks that are directly responsible for toxicity toward cells are still under debate. Here, we have compared the structural characteristics of the toxic intermediate molecular species of αSyn and similar toxic species of another protein, GroES, using coherent X-ray diffraction analysis. Using coherent X-ray free electron laser pulses of SACLA, we analysed αSyn and GroES fibril intermediate species and characterized various aggregate structures. Unlike previous studies where an annular oligomeric form of αSyn was identified, particle reconstruction from scattering traces suggested that the specific forms of the toxic particles were varied, with the sizes of the particles falling within a specific range. We did however discover a common structural feature in both αSyn and GroES samples; the edges of the detected particles were nearly parallel and produced a characteristic diffraction pattern in the diffraction experiments. The presence of parallel-edged particles in toxic intermediates of αSyn and GroES fibrillogenesis pointed towards a plausible common molecular interface that leads to the formation of mature fibrils.

Dynamical Behavior of Human α-Synuclein Studied by Quasielastic Neutron Scattering.

α-synuclein (αSyn) is a protein consisting of 140 amino acid residues and is abundant in the presynaptic nerve terminals in the brain. Although its precise function is unknown, the filamentous aggregates (amyloid fibrils) of αSyn have been shown to be involved in the pathogenesis of Parkinson's disease, which is a progressive neurodegenerative disorder. To understand the pathogenesis mechanism of this disease, the mechanism of the amyloid fibril formation of αSyn must be elucidated. Purified αSyn from bacterial expression is monomeric but intrinsically disordered in solution and forms amyloid fibrils under various conditions. As a first step toward elucidating the mechanism of the fibril formation of αSyn, we investigated dynamical behavior of the purified αSyn in the monomeric state and the fibril state using quasielastic neutron scattering (QENS). We prepared the solution sample of 9.5 mg/ml purified αSyn, and that of 46 mg/ml αSyn in the fibril state, both at pD 7.4 in D2O. The QENS experiments on these samples were performed using the near-backscattering spectrometer, BL02 (DNA), at the Materials and Life Science Facility at the Japan Accelerator Research Complex, Japan. Analysis of the QENS spectra obtained shows that diffusive global motions are observed in the monomeric state but largely suppressed in the fibril state. However, the amplitude of the side chain motion is shown to be larger in the fibril state than in the monomeric state. This implies that significant solvent space exists within the fibrils, which is attributed to the αSyn molecules within the fibrils having a distribution of conformations. The larger amplitude of the side chain motion in the fibril state than in the monomeric state implies that the fibril state is entropically favorable.