Distinct ß-sheet structure in protein aggregates determined by ATR-FTIR spectroscopy.

Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) was used to study the conformation of aggregated proteins in vivo and in vitro. Several different protein aggregates, including amyloid fibrils from several peptides and polypeptides, inclusion bodies, folding aggregates, soluble oligomers, and protein extracts from stressed cells, were examined in this study. All protein aggregates demonstrate a characteristic new ß structure with lower-frequency band positions. All protein aggregates acquire this new ß band following the aggregation process involving intermolecular interactions. The ß sheets in some proteins arise from regions of the polypeptide that are helical or non ß in the native conformation. For a given protein, all types of the aggregates (e.g., inclusion bodies, folding aggregates, and thermal aggregates) showed similar spectra, indicating that they arose from a common partially folded species. All of the aggregates have some nativelike secondary structure and nonperiodic structure as well as the specific new ß structure. The new ß could be most likely attributed to stronger hydrogen bonds in the intermolecular ß-sheet structure present in the protein aggregates.

Oxidized quercetin inhibits α-synuclein fibrillization.

BACKGROUND: α-Synucein is a small (14 kDa), abundant, intrinsically disordered presynaptic protein, whose aggregation is believed to be a critical step in Parkinson's disease (PD). Oxidative stress is reported to be a risk factor for dopamine cell degeneration in PD. Flavonoids are suggested to be important antioxidant against oxidative stress. Flavonoids were reported to inhibit fibrillization and disaggregate the preformed fibrils of α-synucein, but the molecular mechanism was still not clear. METHODS: Quercetin, a well-recognized flavonoid antioxidant, was tested for its inhibition of α-synucein aggregation by thioflavin T assay, light scattering measurement, size-exclusion high performance liquid chromatography, atomic force microscopy, etc. RESULTS: The pre-incubated quercetin exhibited a noticeably stronger inhibition behavior to the fibril formation than that of the freshly prepared. The inhibition is significant in the presence of ortho- and para-benzenediol isomers and inconsiderable in the presence of meta-isomer. The oxidized quercetin species (i.e., chalcantrione, benzyfuranone, quercetinchinone, and other derivatives) cause stronger inhibition than quercetin does because of the elevated polarity and hydrophilicity. Presence of quercetin disaggregates α-synucein fibrils, rather than oligomers and amorphous aggregations. CONCLUSIONS: Instead of the antioxidant activity, the 1:1 covalent binding of quercetin with α-synucein, and the increased hydophilicity of the covalently modified α-synucein oligomers or monomers, account for the inhibition of α-synucein fibrillation. GENERAL SIGNIFICANCE: Clarification of the molecular mechanism of the inhibition and disaggregation may help to screen safer and more effective flavonoid therapeutic in combating PD.

Biophysical Characterization of α-Synuclein and Rotenone Interaction.

Previous studies revealed that pesticides interact with α-synuclein and accelerate the rate of fibrillation. These results are consistent with the prevailing hypothesis that the direct interaction of α-synuclein with pesticides is one of many suspected factors leading to α-synuclein fibrillation and ultimately to Parkinson's disease. In this study, the biophysical properties and fibrillation kinetics of α-synuclein in the presence of rotenone were investigated and, more specifically, the effects of rotenone on the early-stage misfolded forms of α-synuclein were considered. The thioflavine T (ThT) fluorescence assay studies provide evidence that early-phase misfolded α-synuclein forms are affected by rotenone and that the fibrillation process is accelerated. Further characterization by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) shows that rotenone increases the amount of ordered secondary structure in this intrinsically disordered protein. Morphological characterization by transmission electron microscopy (TEM) and atomic force microscopy (AFM) provide visualization of the differences in the aggregated α-synuclein species developing during the early kinetics of the fibrillation process in the absence and presence of rotenone. We believe that these data provide useful information for a better understanding of the molecular basis of rotenone-induced misfolding and aggregation of α-synuclein.

Agrochemicals, α-synuclein, and Parkinson's disease.

Epidemiological, population-based case-control, and experimental studies at the molecular, cellular, and organism levels revealed that exposure to various environmental agents, including a number of structurally different agrochemicals, may contribute to the pathogenesis of Parkinson's disease (PD) and several other neurodegenerative disorders. The role of genetic predisposition in PD has also been increasingly acknowledged, driven by the identification of a number of disease-related genes [e.g., α-synuclein, parkin, DJ-1, ubiquitin C-terminal hydrolase isozyme L1 (UCH-L1), and nuclear receptor-related factor 1]. Therefore, the etiology of this multifactorial disease is likely to involve both genetic and environmental factors. Various neurotoxicants, including agrochemicals, have been shown to elevate the levels of α-synuclein expression in neurons and to promote aggregation of this protein in vivo. Many agrochemicals physically interact with α-synuclein and accelerate the fibrillation and aggregation rates of this protein in vitro. This review analyzes some of the aspects linking α-synuclein to PD, provides brief structural and functional descriptions of this important protein, and represents some data connecting exposure to agrochemicals with α-synuclein aggregation and PD pathogenesis.

Peculiarities of copper binding to alpha-synuclein.

Heavy metals have been implicated as the causative agents for the pathogenesis of the most prevalent neurodegenerative disease. Various mechanisms have been proposed to explain the toxic effects of metals ranging from metal-induced oxidation of protein to metal-induced changes in the protein conformation. Aggregation of a-synuclein is implicated in Parkinson's disease (PD), and various metals, including copper, constitute a prominent group of alpha-synuclein aggregation enhancers. In this study, we have systematically characterized the a-synuclein-Cu21 binding sites and analyzed the possible role of metal binding in a-synuclein fibrillation using a set of biophysical techniques, such as electron paramagnetic resonance (EPR), electron spin-echo envelope modulation (ESEEM), circular dichroism (CD), and size exclusion chromatography (SEC). Our analyses indicated that a-synuclein possesses at least two binding sites for Cu21. We have been able to locate one of the binding sites in the N-terminal region. Furthermore, based on the EPR studies of model peptides and Beta-synuclein, we concluded that the suspected His residue did not appear to participate in strong Cu21 binding.

Conserved C-terminal charge exerts a profound influence on the aggregation rate of α-synuclein.

α-Synuclein (α-syn) is the major component of filamentous Lewy bodies found in the brains of patients diagnosed with Parkinson's disease (PD). Recent studies demonstrate that, in addition to the wild-type sequence, α-syn is found in several modified forms, including truncated and phosphorylated species. Although the mechanism by which the neuronal loss in PD occurs is unknown, aggregation and fibril formation of α-syn are considered to be key pathological features. In this study, we analyze the rates of fibril formation and the monomer-fibril equilibrium for eight disease-associated truncated and phosphorylated α-syn variants. Comparison of the relative rates of aggregation reveals a strong monotonic relationship between the C-terminal charge of α-syn and the lag time prior to the observation of fibril formation, with truncated species exhibiting the fastest aggregation rates. Moreover, we find that a decrease in C-terminal charge shifts the equilibrium to favor the fibrillar species. An analysis of these findings in the context of linear growth theories suggests that the loss of the charge-mediated stabilization of the soluble state is responsible for the enhanced aggregation rate and increased extent of fibril fraction. Therefore, C-terminal charge is kinetically and thermodynamically protective against α-syn polymerization and may provide a target for the treatment of PD.

Characterization of the non-fibrillar α-synuclein oligomers.

Under certain in vitro conditions, α-Synuclein is an abundant 14 kDa presynaptic intrinsically disordered protein, involved in the pathogenesis of Parkinson's disease (PD) forms amyloid fibrils which resemble those found in Lewy bodies of PD patients. However, a substantial fraction of α-synuclein molecules (10-20 %) does not form fibrils during fibrillation and exists in a form of soluble oligomers. In this study, we examined these soluble oligomers by a variety of biophysical techniques including atomic force microscopy (AFM), circular dichroism, Fourier-transform infrared spectroscopy and thioflavin T fluorescence. We observed that the fibrillation kinetics is affected by the variation in salt and protein concentrations. Although both high salt and high protein concentrations noticeably accelerated α-synuclein fibrillation, the amount of non-fibrillar oligomers is independent of the salt content. The oligomers formed at low salt concentration adopt more ß-sheet structure and are smaller in size than those formed at high salt concentration. AFM analysis shows that the low salt oligomers represent a mixture of small oligomers and some amorphous aggregates, whereas oligomers formed at high salt concentrations are noticeably larger, more homogenous, and are mostly spherical in shape. All the late stage non-fibrillar oligomers do not form fibrils even when seeded with pre-formed fibrils, are characterized by negligible rates of dissociation, likely due to their intertwined structure, and are able to disrupt the integrity of the biological membrane. These findings suggest that these soluble oligomers are important players in the multi-pathway aggregation of α-synuclein and should be taken into account in studies on the molecular mechanisms of this protein fibrillation.

Effects of Various Flavonoids on the α-Synuclein Fibrillation Process.

α-Synuclein aggregation and fibrillation are closely associated with the formation of Lewy bodies in neurons and are implicated in the causative pathogenesis of Parkinson's disease and other synucleinopathies. Currently, there is no approved therapeutic agent directed toward preventing the protein aggregation, which has been recently shown to have a key role in the cytotoxic nature of amyloidogenic proteins. Flavonoids, known as plant pigments, belong to a broad family of polyphenolic compounds. Over 4,000 flavonoids have been identified from various plants and foodstuffs derived from plants and have been demonstrated as potential neuroprotective agents. In this study 48 flavonoids belonging to several classes with structures differing in the position of double bonds and ring substituents were tested for their ability to inhibit the fibrillation of α-synuclein in vitro. A variety of flavonoids inhibited α-synuclein fibrillation, and most of the strong inhibitory flavonoids were also found to disaggregate preformed fibrils.

Methionine oxidation stabilizes non-toxic oligomers of alpha-synuclein through strengthening the auto-inhibitory intra-molecular long-range interactions.

Oxidative stress and aggregation of the presynaptic protein alpha-synuclein (alpha-Syn) are implied in the pathogenesis of Parkinson's disease and several other neurodegenerative diseases. Various posttranslational modifications, such as oxidation, nitration and truncation, have significant effects on the kinetics of alpha-Syn fibrillation in vitro. alpha-Syn is a typical natively unfolded protein, which possesses some residual structure. The existence of long-range intra-molecular interactions between the C-terminal tail (residues 120-140) and the central part of alpha-Syn (residues 30-100) was recently established (Bertoncini et al. (2005) Proc Natl Acad Sci U S A 102, 1430-1435). Since alpha-Syn has four methionines, two of which (Met 1 and 5) are at the N-terminus and the other two (Met 116 and 127) are in the hydrophobic cluster at the C-terminus of protein, the perturbation of these residues via their oxidation represents a good model for studying the effect of long-range interaction on alpha-Syn fibril formation. In this paper we show that Met 1, 116, and 127 are more protected from the oxidation than Met 5 likely due to the residual structure in the natively unfolded alpha-Syn. In addition to the hydrophobic interactions between the C-terminal hydrophobic cluster and hydrophobic central region of alpha-Syn, there are some long-range electrostatic interactions in this protein. Both of these interactions likely serve as auto-inhibitors of alpha-Syn fibrillation. Methionine oxidation affects both electrostatic and hydrophobic long-range interactions in alpha-Syn. Finally, oxidation of methionines by H2O2 greatly inhibited alpha-Syn fibrillation in vitro, leading to the formation of relatively stable oligomers, which are not toxic to dopaminergic and GABAergic neurons.

Molecular mechanisms underlying the flavonoid-induced inhibition of alpha-synuclein fibrillation.

The molecular mechanism underlying the flavonoid-induced inhibition of alpha-synuclein fibrillation was thoroughly examined by various biochemical and biophysical approaches. The noncovalent binding of the inhibitory flavonoids to alpha-synuclein and the covalent modification by the flavonoid quinone led to the restriction of the conformational changes in this natively unfolded protein and to the stabilization of soluble flavonoid-modified species of alpha-synuclein (monomers and oligomers). All of these factors rather than a single one contribute to the inhibition of WT alpha-synuclein fibrillation induced by the flavonoid. The structural requirements that appear necessary to provide a flavonoid the ability to inhibit alpha-synuclein fibrillation were determined to be vicinal dihydroxyphenyl moieties, irrespective of the ring position where they are located. Flavonoids with three vicinal hydroxyl groups exhibited enhanced inhibitory effects on alpha-synuclein fibrillation. The antioxidant activities of flavonoids were generally correlated with their in vitro inhibitory effects on alpha-synuclein fibrillation. The flavonoids inhibiting alpha-synuclein fibrillation and stabilizing the protein monomeric conformation can serve as a model for the development of therapeutic drugs in combating Parkinson's disease.

At low concentrations, 3,4-dihydroxyphenylacetic acid (DOPAC) binds non-covalently to alpha-synuclein and prevents its fibrillation.

Several studies have shown that catecholamines can inhibit the fibrillation of alpha-synuclein (alpha-Syn), a small presynaptic protein whose aggregation is believed to be a critical step in the etiology of Parkinson's disease and several other neurodegenerative disorders. However, the mechanism of this inhibition is uncertain. We show here that substoichiometric concentrations of 3,4-dihydroxyphenylacetic acid (DOPAC), a normal product of the metabolism of dopamine, can inhibit the fibrillation of alpha-Syn, due to non-covalent binding of DOPAC to alpha-Syn monomer. Intriguingly, the presence of alpha-Syn accelerates the spontaneous oxidation of DOPAC, and the oxidized form of DOPAC (the quinone) is responsible for the fibrillation inhibition. In addition, the presence of DOPAC leads to the oxidation of the methionine residues of alpha-Syn, probably due to the H(2)O(2) production as a by-product of DOPAC oxidation. The lack of fibrillation results from the formation of stable oligomers, which are very similar to those observed transiently at early stages of the alpha-Syn fibrillation. A possible explanation for this phenomenon is that DOPAC stabilizes the normally transient oligomers and prevents them from subsequent fibril formation. The analysis of the alpha-Syn Y39W variant suggests that DOPAC binds non-covalently to the same N-terminal region of alpha-Syn as lipid vesicles, probably in the vicinity of residue 39. In contrast to the compounds with 1,2-dihydroxyphenyl groups (DOPAC and catechol), their 1,4-dihydroxyphenyl isomers (hydroquinone and homogentisic acid) are able to modify alpha-Syn covalently, probably due to the less steric hindrance in the Michael addition.

Smoking and Parkinson's disease: does nicotine affect alpha-synuclein fibrillation?

alpha-synuclein is a small presynaptic protein (14,460 D) that is abundantly distributed in the brain. Although, its function is unknown, the aggregated form of alpha-synuclein is a pathological hallmark of several neurodegenerative diseases, including Parkinson's disease (PD). Epidemiological studies have shown that smoking can lessen the incidence of Parkinson's disease, indicating that smoke may contain chemicals that are neuro-protective. The fibrillation of alpha-synuclein was studied in relation to five different compounds found in cigarette smoke: anabasine, cotinine, hydroquinone, nicotine and nornicotine. Thioflavin T assays, gel electrophoresis, size exclusion chromatography-high performance liquid chromatography (SEC-HPLC) and atomic force microscopy (AFM) were utilized to monitor the rate of alpha-synuclein fibrillation and the inhibitory effects of the cigarette smoke components. We show that nicotine and hydroquinone inhibit alpha-synuclein fibril formation in a concentration-dependent manner, with nicotine being more effective. The SEC-HPLC data show that nicotine and hydroquinone stabilize soluble oligomers. The morphology of the oligomers stabilized by nicotine was evaluated by AFM, which showed the presence of three stable oligomers with an average height of 16 nm, 10 nm and 4 nm. Comparable results were obtained for the effect of the cigarette smoke components on the A53T mutant fibrillation. These results show that nicotine and hydroquinone inhibit alpha-synuclein fibrillation and stabilize soluble oligomeric forms. This information can be used to understand the molecular mechanism of the nicotine and hydroquinone action to develop therapeutic solutions for PD.

Concerted action of metals and macromolecular crowding on the fibrillation of alpha-synuclein.

Certain metals lead to increased risk of Parkinson's disease (PD) and the aggregation of alpha-synuclein is implicated in the PD pathology. Although alpha-synuclein fibrillation has been extensively studied in dilute solutions in vitro, the intracellular environment is highly crowded. We are showing here that certain metals cause a significant acceleration of alpha-synuclein fibrillation in the presence of high concentrations of various macromolecules mostly through decreasing the fibrillation lagtime. The faster fibrillation in crowded environments in the presence of heavy metals suggests a simple molecular basis for the observed elevated risk of PD due to exposure to metals.

Structural characteristics of alpha-synuclein oligomers stabilized by the flavonoid baicalein.

The flavonoid baicalein inhibits fibrillation of alpha-synuclein, which is a major component of Lewy bodies in Parkinson's disease. It has been known that baicalein induces the formation of alpha-synuclein oligomers and consequently prevents their fibrillation. In order to evaluate the structural properties of baicalein-stabilized oligomers, we purified oligomer species by HPLC and examined their stability and structure by CD, Fourier transform infrared spectroscopy, size exclusion chromatography HPLC, small-angle X-ray scattering, and atomic force microscopy. Baicalein-stabilized oligomers are beta-sheet-enriched according to CD and Fourier transform infrared spectroscopy analyses. They did not form fibrils even after very prolonged incubation. From small-angle X-ray scattering data and atomic force microscopy images, the oligomers were characterized as quite compact globular species. Oligomers were extremely stable, with a GdmCl C(m)=3.3 M. This high stability explains the previously observed inhibition properties of baicalein against alpha-synuclein fibrillation. These baicalein-stabilized oligomers, added to the solution of aggregating alpha-synuclein, were able to noticeably inhibit its fibrillation. After prolonged coincubation, short fibrils were formed, suggesting an effective interaction of oligomers with monomeric alpha-synuclein. Membrane permeability tests suggested that the baicalein-stabilized oligomers had a mild effect on the integrity of the membrane surface. This effect was rather similar to that of the monomeric protein, suggesting that targeted stabilization of certain alpha-synuclein oligomers might offer a potential strategy for the development of novel Parkinson's disease therapies.

Impact of Tyr to Ala mutations on alpha-synuclein fibrillation and structural properties.

Substantial evidence suggests that the fibrillation of alpha-synuclein is a critical step in the development of Parkinson's disease. In vitro, alpha-synuclein forms fibrils with morphologies and a staining characteristic similar to those extracted from disease-affected brain. Monomeric alpha-synuclein is an intrinsically disordered protein, with three Tyr residues in the C-terminal region, one in the N-terminus, and lacking Trp. It is thought that interactions between the C-terminus and the central portion of the molecule may prevent or minimize aggregation/fibrillation. To test this hypothesis we examined the importance of the Tyr residues on the propensity for alpha-synuclein to fibrillate in vitro. Fibril formation of alpha-synuclein was completely inhibited, in the timescale over which measurements were made, by replacing the three C-terminal Tyr residues with Ala. In addition, substitution of Tyr133 by Ala also resulted in the absence of fibrillation, whereas the individual Y125A and Y136A mutants showed limited inhibition. Replacement of Tyr39 by Ala also resulted in substantial inhibition of fibrillation. Structural analysis showed that the Y133A mutant had a substantially different conformation, rich in alpha-helical secondary structure, as compared with the wild-type and other mutants, although the formation of any tertiary structure has not been observed as can be judged from near-UV-CD spectra. These observations suggest that the long-range intramolecular interactions between the N- and C-termini of alpha-synuclein are likely to be crucial to the fibrillation process.

The effect of membranes on the in vitro fibrillation of an amyloidogenic light-chain variable-domain SMA.

Light chain (or AL) amyloidosis is the most common form of systemic amyloidosis, characterized by the pathological deposition of insoluble fibrils of immunoglobulin light-chain fragments in various organs and tissues, especially in the kidney and heart. Both the triggering factors and the mechanisms involved in the abnormal formation of the insoluble fibrillar aggregates from the soluble proteins are poorly understood. For example, although the fibrillar deposits are typically found associated with the extracellular matrix and basement membranes, it is not clear whether fibrils are initially formed intra- or extracellularly, nor it is understood what determines where the deposits will occur; i.e., site tropism. In the present investigation, we studied the interaction of a recombinant amyloidogenic light-chain variable domain, SMA, with lipid vesicles. The nature of the interaction was dependent on the lipid composition and the SMA to lipid ratio. The most pronounced effect was found from vesicles composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate, which dramatically accelerated fibril growth. Interestingly, spectral probes, such as intrinsic fluorescence and far-UV CD spectroscopy did not show significant conformational changes in the presence of the vesicles. The presence of cholesterol or divalent cations, such as Ca(2+) and Mg(2+), lead to decreased membrane-induced SMA fibrillation. Thus, membranes may have significant effects on light-chain fibrillation and may contribute to the site selectivity observed in AL amyloidosis.

Guiding protein aggregation with macromolecular crowding.

Macromolecular crowding is expected to have a significant effect on protein aggregation. In the present study we analyzed the effect of macromolecular crowding on fibrillation of four proteins, bovine S-carboxymethyl-alpha-lactalbumin (a disordered form of the protein with reduced three out of four disulfide bridges), human insulin, bovine core histones, and human alpha-synuclein. These proteins are structurally different, varying from natively unfolded (alpha-synuclein and core histones) to folded proteins with rigid tertiary and quaternary structures (monomeric and hexameric forms of insulin). All these proteins are known to fibrillate in diluted solutions, however their aggregation mechanisms are very divers and some of them are able to form different aggregates in addition to fibrils. We studied how macromolecular crowding guides protein between different aggregation pathways by analyzing the effect of crowding agents on the aggregation patterns under the variety of conditions favoring different aggregated end products in diluted solutions.

Effect of methionine oxidation on the structural properties, conformational stability, and aggregation of immunoglobulin light chain LEN.

Light chain amyloidoses arise from the overproduction and abnormal deposition of the immunoglobulin light chain in various organs. LEN is the variable domain of an immunoglobulin light chain originally isolated from the urine of a patient suffering from multiple myeloma, with no sign of renal dysfunction or amyloidosis. LEN was shown to form fibrils in vitro under mildly destabilizing conditions. In this work, we investigated the changes induced by methionine oxidation in the structural properties, conformational stability, and aggregation behavior of immunoglobulin light chain domain LEN. We established that LEN was well-protected from oxidation in its native state, but successful oxidation was achieved in the presence of 4 M GuHCl. Oxidation induced noticeable structural changes in LEN and destabilized this protein. The methionine-oxidized LEN preferred to form amorphous aggregates instead of fibrils. The results indicated that the LEN oxidation may play an important role in amorphous deposition of the protein, but not in its fibrillation.

Structural impact of three Parkinsonism-associated missense mutations on human DJ-1.

A number of missense mutations in the oxidative stress response protein DJ-1 are implicated in rare forms of familial Parkinsonism. The best-characterized Parkinsonian DJ-1 missense mutation, L166P, disrupts homodimerization and results in a poorly folded protein. The molecular basis by which the other Parkinsonism-associated mutations disrupt the function of DJ-1, however, is incompletely understood. In this study we show that three different Parkinsonism-associated DJ-1 missense mutations (A104T, E163K, and M26I) reduce the thermal stability of DJ-1 in solution by subtly perturbing the structure of DJ-1 without causing major folding defects or loss of dimerization. Atomic resolution X-ray crystallography shows that the A104T substitution introduces water and a discretely disordered residue into the core of the protein, E163K disrupts a key salt bridge with R145, and M26I causes packing defects in the core of the dimer. The deleterious effect of each Parkinsonism-associated mutation on DJ-1 is dissected by analysis of engineered substitutions (M26L, A104V, and E163K/R145E) that partially alleviate each of the defects introduced by the A104T, E163K and M26I mutations. In total, our results suggest that the protective function of DJ-1 can be compromised by diverse perturbations in its structural integrity, particularly near the junctions of secondary structural elements.

Role of different regions of alpha-synuclein in the assembly of fibrils.

Elucidating the details of the assembly of amyloid fibrils is a key step to understanding the mechanism of amyloid deposition diseases including Parkinson's disease. Although several models have been proposed, based on analyses of polypeptides and short peptides, a detailed understanding of the structure and mechanism of alpha-synuclein fibrillation remains elusive. In this study, we used trypsin and endoproteinase GluC to digest intact alpha-synuclein fibrils and to analyze the detailed morphology of the resultant fibrils/remnants. We also created three mutants of alpha-synuclein, in which the N-terminal and C-terminal regions were removed, both individually and in combination, and investigated the detailed morphology of the fibrils from these mutants. Our results indicate that the assembly of mature alpha-synuclein fibrils is hierarchical: protofilaments --> protofibrils --> mature fibrils. There is a core region of approximately 70 amino acids, from residues approximately 32 to 102, which comprises the beta-rich core of the protofilaments and fibrils. In contrast, the two terminal regions show no evidence of participating in the assembly of the protofilament core but play a key role in the interactions between the protofilaments, which is necessary for the fibril maturation.