Structure and texture of fibrous crystals formed by Alzheimer's abeta(11-25) peptide fragment

Amyloid fibril deposition is central to the pathology of Alzheimer's disease. X-ray diffraction from amyloid fibrils formed from full-length Abeta(1-40) and from a shorter fragment, Abeta(11-25), have revealed cross-beta diffraction fingerprints. Magnetic alignment of Abeta(11-25) amyloid fibrils gave a distinctive X-ray diffraction texture, allowing interpretation of the diffraction data and a model of the arrangement of the peptides within the amyloid fiber specimen to be constructed. An intriguing feature of the structure of fibrillar Abeta(11-25) is that the beta sheets, of width 5.2 nm, stack by slipping relative to each other by the length of two amino acid units (0.70 nm) to form beta ribbons 4.42 nm in thickness. Abeta(1-40) amyloid fibrils likely consist of once-folded hairpins, consistent with the size of the fibers obtained using electron microscopy and X-ray diffraction.     

The circularization of amyloid fibrils formed by apolipoprotein C-II

Amyloid fibrils have historically been characterized by diagnostic dye-binding assays, their fibrillar morphology, and a "cross-beta" x-ray diffraction pattern. Whereas the latter demonstrates that amyloid fibrils have a common beta-sheet core structure, they display a substantial degree of morphological variation. One striking example is the remarkable ability of human apolipoprotein C-II amyloid fibrils to circularize and form closed rings. Here we explore in detail the structure of apoC-II amyloid fibrils using electron microscopy, atomic force microscopy, and x-ray diffraction studies. Our results suggest a model for apoC-II fibrils as ribbons approximately 2.1-nm thick and 13-nm wide with a helical repeat distance of 53 nm +/- 12 nm. We propose that the ribbons are highly flexible with a persistence length of 36 nm. We use these observed biophysical properties to model the apoC-II amyloid fibrils either as wormlike chains or using a random-walk approach, and confirm that the probability of ring formation is critically dependent on the fibril flexibility. More generally, the ability of apoC-II fibrils to form rings also highlights the degree to which the common cross-beta superstructure can, as a function of the protein constituent, give rise to great variation in the physical properties of amyloid fibrils.     

Effect of environmental conditions on aggregation and fibril formation of barstar

The dependence on environmental conditions of the assembly of barstar into amyloid fibrils was investigated starting from the nonnative, partially folded state at low pH (A-state). The kinetics of this process was monitored by CD spectroscopy and static and dynamic light scattering. The morphology of the fibrils was visualized by electron microscopy, while the existence of the typical cross- structure substantiated by solution X-ray scattering. At room temperature, barstar in the A-state is unable to form amyloid fibrils, instead amorphous aggregation is observed at high ionic strength. Further destabilization of the structure is required to transform the polypeptide chain into an ensemble of conformations capable of forming amyloid fibrils. At moderate ionic strength (75 mM NaCl), the onset and the rate of fibril formation can be sensitively tuned by increasing the temperature. Two types of fibrils can be detected differing in their morphology, length distribution and characteristic far UV CD spectrum. The formation of the different types depends on the particular environmental conditions. The sequence of conversion: A-statefibril type Ifibril type II appears to be irreversible. The transition into fibrils is most effective when the protein chain fulfills particular requirements concerning secondary structure, structural flexibility and tendency to cluster.Abbreviations CD circular dichroism - DLS dynamic light scattering - EM electron microscopy - SLS static light scattering - SAXS small-angle X-ray scattering - SOXS solution X-ray scattering     

Glycation induces formation of amyloid cross-beta structure in albumin

Amyloid fibrils are components of proteinaceous plaques that are associated with conformational diseases such as Alzheimer's disease, transmissible spongiform encephalopathies, and familial amyloidosis. Amyloid polypeptides share a specific quarternary structure element known as cross-beta structure. Commonly, fibrillar aggregates are modified by advanced glycation end products (AGE). In addition, AGE formation itself induces protein aggregation. Both amyloid proteins and protein-AGE adducts bind multiligand receptors, such as receptor for AGE, CD36, and scavenger receptors A and B type I, and the serine protease tissue-type plasminogen activator (tPA). Based on these observations, we hypothesized that glycation induces refolding of globular proteins, accompanied by formation of cross-beta structure. Using transmission electron microscopy, we demonstrate here that glycated albumin condensates into fibrous or amorphous aggregates. These aggregates bind to amyloid-specific dyes Congo red and thioflavin T and to tPA. In contrast to globular albumin, glycated albumin contains amino acid residues in beta-sheet conformation, as measured with circular dichroism spectropolarimetry. Moreover, it displays cross-beta structure, as determined with x-ray fiber diffraction. We conclude that glycation induces refolding of initially globular albumin into amyloid fibrils comprising cross-beta structure. This would explain how glycated ligands and amyloid ligands can bind to the same multiligand "cross-beta structure" receptors and to tPA.     

Kinetics of aggregation of synthetic beta-amyloid peptide.

beta-Amyloid peptide is the major protein component of senile plaques and cerebrovascular amyloid deposits in patients with Alzheimer's disease. The peptide deposits extracellularly in the form of amyloid fibrils, in a cross-beta conformation. beta-amyloid peptide is a 39- to 43-residue segment of a normal membrane precursor protein. In this work, a peptide homologous to the first 40 amino acids of beta-amyloid peptide, beta(1-40), was synthesized and characterized. beta(1-40) exhibited a sharp change in solubility near physiological pH and gel formation at concentrations of 3 mg/ml or greater. Circular dichroism indicated that beta(1-40) contained approximately two-thirds beta-structure, but no alpha-helical character. Quasi-elastic and classical light scattering measurements showed that beta(1-40) aggregated end-to-end in solution, reaching average molecular weights greater than 4 x 10(6) after 13 days. The aggregates were best modeled as rigid rods of 5 nm diameter, similar to the diameter of amyloid fibrils purified from plaques. A mathematical model based on diffusion-limited aggregation was developed to describe the kinetics of aggregation.     

Inflammation protein SAA2.2 spontaneously forms marginally stable amyloid fibrils at physiological temperature

For nearly four decades, the formation of amyloid fibrils by the inflammation-related protein serum amyloid A (SAA) has been pathologically linked to the disease amyloid A (AA) amyloidosis. However, here we show that the nonpathogenic murine SAA2.2 spontaneously forms marginally stable amyloid fibrils at 37 °C that exhibit cross-beta structure, binding to thioflavin T, and fibrillation by a nucleation-dependent seeding mechanism. In contrast to the high stability of most known amyloid fibrils to thermal and chemical denaturation, experiments monitored by glutaraldehyde cross-linking/SDS-PAGE, thioflavin T fluorescence, and light scattering (OD(600)) showed that the mature amyloid fibrils of SAA2.2 dissociate upon incubation in >1.0 M urea or >45 °C. When considering the nonpathogenic nature of SAA2.2 and its ~1000-fold increased concentration in plasma during an inflammatory response, its extreme in vitro amyloidogenicity under physiological-like conditions suggest that SAA amyloid might play a functional role during inflammation. Of general significance, the combination of methods used here is convenient for exploring the stability of amyloid fibrils that are sensitive to urea and temperature. Furthermore, our studies imply that analogous to globular proteins, which can possess structures ranging from intrinsically disordered to extremely stable, amyloid fibrils formed in vivo might have a broader range of stabilities than previously appreciated with profound functional and pathological implications.     

Alpha-helix structure in Alzheimer's disease aggregates of tau-protein

The discovery of beta-sheet structure in Alzheimer's amyloid fibrils, and then in many other disease-related protein fibrils, has led to the widely believed view that beta-sheet formation is the general mechanism of aberrant protein aggregation leading to disease. This notion is further reinforced by recent findings, which indicate that normal proteins can be induced to form beta-sheet fibrils in vitro. Alzheimer's disease, a paradigm proteopathy, is accompanied by the formation of two distinct aggregates, amyloid fibrils and paired helical filaments (PHFs). Electron microscope images of PHFs show pairs of twisted ribbons with 80 nm periodicity. However, there is little information of the molecular structure of PHFs, as previous studies have failed to identify signs of regular structure. Using far-UV circular dichroism and Fourier-transformed infrared spectroscopy, we find that PHFs are comprised of alpha-helices. This is remarkable as tau-protein, PHF's primary constituent, has a high abundance of helix-breaking amino acids and is unstructured in solution. We also find that PHFs are very stable, as judged by their high melting temperature and resistance to protease digestion. PHFs are the first example of pathological aggregation associated to the formation of alpha-helix.     

Amyloid fibrils derived from the apolipoprotein A1 Leu174Ser variant contain elements of ordered helical structure

We recently described a new apolipoprotein A1 variant presenting a Leu174Ser replacement mutation that is associated with a familial form of systemic amyloidosis displaying predominant heart involvement. We have now identified a second unrelated patient with very similar clinical presentation and carrying the identical apolipoprotein A1 mutation. In this new patient the main protein constituent of the amyloid fibrils is the polypeptide derived from the first 93 residues of the protein, the identical fragment to that found in the patient previously described to carry this mutation. The X-ray fiber diffraction pattern obtained from preparations of partially aligned fibrils displays the cross-beta reflections characteristic of all amyloid fibrils. In addition to these cross-beta reflections, other reflections suggest the presence of well-defined coiled-coil helical structure arranged with a defined orientation within the fibrils. In both cases the fibrils contain a trace amount of full-length apolipoprotein A1 with an apparent prevalence of the wild-type species over the variant protein. We have found a ratio of full-length wild-type to mutant protein in plasma HDL of three to one. The polypeptide 1--93 purified from natural fibrils can be solubilized in aqueous solutions containing denaturants, and after removal of denaturants it acquires a monomeric state that, based on CD and NMR studies, has a predominantly random coil structure. The addition of phospholipids to the monomeric form induces the formation of some helical structure, thought most likely to occur at the C-terminal end of the polypeptide.     

An Insight into the pathway of the amyloid fibril formation of hen egg white lysozyme obtained from a small-angle X-ray and neutron scattering study

It is known that hen egg white lysozyme (HEWL) forms amyloid fibrils. Since HEWL is one of the proteins that have been studied most extensively and is closely related to human lysozyme, the variants of which form the amyloid fibrils that are related to hereditary systemic amyloidosis, this protein is an ideal model to study the mechanism of amyloid fibril formation. In order to gain an insight into the mechanism of amyloid fibril formation, systematic and detailed studies to detect and characterize various structural states of HEWL were conducted. Since HEWL forms amyloid fibrils in highly concentrated ethanol solutions, solutions of various concentrations of HEWL in various concentrations of ethanol were prepared, and the structures of HEWL in these solutions were investigated by small-angle X-ray and neutron scattering. It was shown that the structural states of HEWL were distinguished as the monomer state, the state of the dimer formation, the state of the protofilament formation, the protofilament state, and the state towards the formation of amyloid fibrils. A phase diagram of these structural states was obtained as a function of protein, water and ethanol concentrations. It was found that under the monomer state the structural changes of HEWL were not gross changes in shape but local conformational changes, and the dimers, formed by the association at the end of the long axis of HEWL, had an elongated shape. Circular dichroism measurements showed that the large changes in the secondary structures of HEWL occurred during dimer formation. The protofilaments were formed by stacking of the dimers with their long axis (nearly) perpendicular to and rotated around the protofilament axis to form a helical structure. These protofilaments were characterized by their radius of gyration of the cross-section of 2.4nm and the mass per unit length of 16,000(+/-2300)Da/nm. It was shown that the changes of the structural states towards the amyloid fibril formation occurred via lateral association of the protofilaments. A pathway of the amyloid fibril formation of HEWL was proposed from these results.     

The process of amyloid-like fibril formation by methionine aminopeptidase from a hyperthermophile, Pyrococcus furiosus

Amyloid is associated with serious diseases including Alzheimer's disease and senile-systemic amyloidosis due to misfolded proteins. In the course of study of the denaturation process of methionine aminopeptidase (MAP) from the hyperthermophile P. furiosus, we found that MAP forms amyloid-like fibrils, and we then investigated the mechanism of amyloid fibril formation. The kinetic experiments on denaturation monitored by CD at 222 nm indicated that MAP in the presence of 3.37 M GuHCl at pH 3.31 changed to a conformation containing a considerable content of beta-sheet structure after the destruction of the alpha-helical structure. MAP in this beta-rich conformation was highly associated, and its stability was remarkably high: the midpoint of the GuHCl denaturation curve was 4.82 M at pH 3.0, and a thermal transition was not observed up to 125 degrees C by calorimetry. The amyloid-like fibril formation of MAP was confirmed by Congo red staining with a typical peak at 542 nm in the difference spectrum, showing a cross-beta X-ray diffraction pattern with a clear sharp reflection at 4.7 A and a characteristic unbranched fibrillar appearance with a length of about 1000 A and a diameter of about 70 A in the electron micrographs. Present results indicate that the amyloid-like form of MAP appears just after the protein is almost completely denatured, and even highly stable proteins can also form amyloid-like conformation under conditions where the denatured state of the protein is abundantly populated.     

Bacterial inclusion bodies contain amyloid-like structure

Protein aggregation is a process in which identical proteins self-associate into imperfectly ordered macroscopic entities. Such aggregates are generally classified as amorphous, lacking any long-range order, or highly ordered fibrils. Protein fibrils can be composed of native globular molecules, such as the hemoglobin molecules in sickle-cell fibrils, or can be reorganized beta-sheet-rich aggregates, termed amyloid-like fibrils. Amyloid fibrils are associated with several pathological conditions in humans, including Alzheimer disease and diabetes type II. We studied the structure of bacterial inclusion bodies, which have been believed to belong to the amorphous class of aggregates. We demonstrate that all three in vivo-derived inclusion bodies studied are amyloid-like and comprised of amino-acid sequence-specific cross-beta structure. These findings suggest that inclusion bodies are structured, that amyloid formation is an omnipresent process both in eukaryotes and prokaryotes, and that amino acid sequences evolve to avoid the amyloid conformation.     

A systematic investigation into the effect of protein destabilisation on beta 2-microglobulin amyloid formation

Beta-2-microglobulin (beta(2)m) has been shown to form amyloid fibrils with distinct morphologies under acidic conditions in vitro. Short, curved fibrils (<600 nm in length), form rapidly without a lag phase, with a maximum rate at pH 3.5. By contrast, fibrils with a long (approximately 1 microm), straight morphology are produced by incubation of the protein at pH< or =3.0. Both fibril types display Congo red birefringence, bind Thioflavin-T and have X-ray fibre diffraction patterns consistent with a cross-beta structure. In order to investigate the role of different partially folded states in generating fibrils of each type, and to probe the effect of protein stability on amyloid formation, we have undertaken a detailed mutagenesis study of beta(2)m. Thirteen variants containing point mutations in different regions of the native protein were created and their structure, stability and fibril forming propensities were investigated as a function of pH. By altering the stability of the native protein in this manner, we show that whilst destabilisation of the native state is important in the generation of amyloid fibrils, population of specific denatured states is a pre-requisite for amyloid formation from this protein. Moreover, we demonstrate that the formation of fibrils with different morphologies in vitro correlates with the relative population of different precursor states.     

The native-like conformation of Ure2p in fibrils assembled under physiologically relevant conditions switches to an amyloid-like conformation upon heat-treatment of the fibrils

The [URE3] phenotype in the yeast Saccharomyces cerevisiae is inherited by a prion mechanism involving self-propagating Ure2p aggregates. It is believed that assembly of intact Ure2p into fibrillar polymers that bind Congo Red and show yellow-green birefringence upon staining and are resistant to proteolysis is the consequence of a major change in the conformation of the protein. We recently dissected the assembly process of Ure2p and showed the protein to retain its native alpha-helical structure upon assembly into protein fibrils that are similar to amyloids in that they are straight, bind Congo red and show green-yellow birefringence and have an increased resistance to proteolysis (). Here we further show using specific ligand binding, FTIR spectroscopy and X-ray fiber diffraction that Ure2p fibrils assembled under physiologically relevant conditions are devoid of a cross-beta core. The X-ray fiber diffraction pattern of these fibrils reveals their well-defined axial supramolecular order. By analyzing the effect of heat-treatment on Ure2p fibrils we bring evidences for a large conformational change that occurs within the fibrils with the loss of the ligand binding capacity, decrease of the alpha helicity, the formation of a cross-beta core and the disappearance of the axial supramolecular order. The extent of the conformational change suggests that it is not limited to the N-terminal part of Ure2p polypeptide chain. We show that the heat-treated fibrils that possess a cross-beta core are unable to propagate their structural characteristic while native-like fibrils are. Finally, the potential evolution of native-like fibrils into amyloid fibrils is discussed.     

Infrared linear dichroism spectroscopy on amyloid fibrils aligned by molecular combing

We report the use of molecular combing as an alignment method to obtain macroscopically oriented amyloid fibrils on planar surfaces. The aligned fibrils are studied by polarized infrared spectroscopy. This gives structural information that cannot be definitively obtained from standard infrared experiments on isotropic samples, for example, confirmation of the characteristic cross-β amyloid core structure, the side-chain orientation from specific amino acids, and the arrangement of the strands within the fibrils, as we demonstrate here. We employed amyloid fibrils from hen egg white lysozyme (HEWL) and from a model octapeptide. Our results demonstrate molecular combing as a straightforward method to align amyloid fibrils, producing highly anisotropic infrared linear dichroism (IRLD) spectra.     

Amyloid fibril formation from sequences of a natural beta-structured fibrous protein, the adenovirus fiber

Amyloid fibrils are fibrous beta-structures that derive from abnormal folding and assembly of peptides and proteins. Despite a wealth of structural studies on amyloids, the nature of the amyloid structure remains elusive; possible connections to natural, beta-structured fibrous motifs have been suggested. In this work we focus on understanding amyloid structure and formation from sequences of a natural, beta-structured fibrous protein. We show that short peptides (25 to 6 amino acids) corresponding to repetitive sequences from the adenovirus fiber shaft have an intrinsic capacity to form amyloid fibrils as judged by electron microscopy, Congo Red binding, infrared spectroscopy, and x-ray fiber diffraction. In the presence of the globular C-terminal domain of the protein that acts as a trimerization motif, the shaft sequences adopt a triple-stranded, beta-fibrous motif. We discuss the possible structure and arrangement of these sequences within the amyloid fibril, as compared with the one adopted within the native structure. A 6-amino acid peptide, corresponding to the last beta-strand of the shaft, was found to be sufficient to form amyloid fibrils. Structural analysis of these amyloid fibrils suggests that perpendicular stacking of beta-strand repeat units is an underlying common feature of amyloid formation.     

Filaments of the Ure2p prion protein have a cross-beta core structure

Formation of filaments by the Ure2 protein constitutes the molecular mechanism of the [URE3] prion in yeast. According to the "amyloid backbone" model, the N-terminal asparagine-rich domains of Ure2p polymerize to form an amyloid core fibril that is surrounded by C-terminal domains in their native conformation. Protease resistance and Congo Red binding as well as beta-sheet content detected by spectroscopy-all markers for amyloid-have supported this model, as has the close resemblance between 40 A N-domain fibrils and the fibrillar core of intact Ure2p filaments visualized by cryo-electron microscopy and scanning transmission electron microscopy. Here, we present electron diffraction and X-ray diffraction data from filaments of Ure2p, of N-domains alone, of fragments thereof, and of an N-domain-containing fusion protein that demonstrate in each case the 4.7A reflection that is typical for cross-beta structure and highly indicative of amyloid. This reflection was observed for specimens prepared by air-drying with and without sucrose embedding. To confirm that the corresponding structure is not an artifact of air-drying, the reflection was also demonstrated for specimens preserved in vitreous ice. Local area electron diffraction and X-ray diffraction from partially aligned specimens showed that the 4.7A reflection is meridional and therefore the underlying structure is cross-beta.     

The parallel superpleated beta-structure as a model for amyloid fibrils of human amylin

Human amylin is a 37 amino acid residue peptide hormone whose fibrillogenesis has been correlated with type 2 diabetes. These fibrils are rope-like bundles of several 5nm diameter protofilaments. Here, we propose, as a model for the protofilament, a variant of the parallel superpleated beta-structure previously derived for amyloid filaments of the yeast prion Ure2p. In the amylin model, individual polypeptides from residues 9 to 37 have a planar S-shaped fold with three beta-strands. These serpentines are stacked in register, with a 0.47 nm axial rise and a small rotational twist per step, generating an array of three parallel beta-sheets in cross-beta conformation. The interior, the two "bays" sandwiched between adjacent sheets, are occupied by non-polar and by polar/uncharged residues that are predicted to form H-bonded ladders, similar to those found in beta-helical proteins. The N-terminal peptide containing a disulfide bond occupies an extraneous peripheral position in the protofilament. The left-handed twist of the beta-sheets is shown to underlie left-handed coiling of amylin protofilaments in fibrils. The model is consistent with current biophysical, biochemical and genetic data and, in particular, affords a plausible explanation for why rodent amylin does not form fibrils.     

A single mutation induces amyloid aggregation in the alpha-spectrin SH3 domain: analysis of the early stages of fibril formation

The Src-homology region 3 domain of chicken alpha-spectrin (Spc-SH3) is a small two-state folding protein, which has never been described to form amyloid fibrils under any condition investigated so far. We show here that the mutation of asparagine 47 to alanine at the distal loop, which destabilises similarly the native and folding transition states of the domain, induces the formation of amyloid fibrils under mild acid conditions. Amyloid aggregation of the mutant is enhanced by the increase in temperature, protein concentration and NaCl concentration. The early stages of amyloid formation have been monitored as a function of time and temperature using a variety of biophysical methods. Differential scanning calorimetry experiments under conditions of amyloid formation have allowed the identification of different thermal transitions corresponding to conformational and aggregation processes as well as to the high-temperature disaggregation and unfolding of the amyloid fibrils. Aggregation is preceded by a rapid conformational change in the monomeric domain involving about 40% of the global unfolding enthalpy, considerable change in secondary structure, large loss of tertiary structure and exposure of hydrophobic patches to the solvent. The conformational change is followed by formation of a majority of oligomeric species with apparent hydrodynamic radius between 2.5 nm and 10nm, depending on temperature, together with the appearance and progressive growth of protofibrillar aggregates. After these early aggregation stages, long and curved fibrils of up to several micrometers start to develop by elongation of the protofibrils. The calorimetric data indicate that the specific enthalpy of fibril disaggregation and unfolding is relatively low, suggesting a low density of interactions within the fibril structure as compared to the native protein and a main entropy contribution to the stability of the amyloid fibrils.     

A Simple Lattice Model That Captures Protein Folding,Aggregation and Amyloid Formation

The ability of many proteins to convert from their functional soluble state to amyloid fibrils can be attributed to inter-molecular beta strand formation. Such amyloid formation is associated with neurodegenerative disorders like Alzheimer''s and Parkinson''s. Molecular modelling can play a key role in providing insight into the factors that make proteins prone to fibril formation. However, fully atomistic models are computationally too expensive to capture the length and time scales associated with fibril formation. As the ability to form fibrils is the rule rather than the exception, much insight can be gained from the study of coarse-grained models that capture the key generic features associated with amyloid formation. Here we present a simple lattice model that can capture both protein folding and beta strand formation. Unlike standard lattice models, this model explicitly incorporates the formation of hydrogen bonds and the directionality of side chains. The simplicity of our model makes it computationally feasible to investigate the interplay between folding, amorphous aggregation and fibril formation, and maintains the capability of classic lattice models to simulate protein folding with high specificity. In our model, the folded proteins contain structures that resemble naturally occurring beta-sheets, with alternating polar and hydrophobic amino acids. Moreover, fibrils with intermolecular cross-beta strand conformations can be formed spontaneously out of multiple short hydrophobic peptide sequences. Both the formation of hydrogen bonds in folded structures and in fibrils is strongly dependent on the amino acid sequence, indicating that hydrogen-bonding interactions alone are not strong enough to initiate the formation of beta sheets. This result agrees with experimental observations that beta sheet and amyloid formation is strongly sequence dependent, with hydrophobic sequences being more prone to form such structures. Our model should open the way to a systematic study of the interplay between the factors that lead to amyloid formation.     

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

The flavonoid baicalein inhibits fibrillation of α-synuclein, which is a major component of Lewy bodies in Parkinson's disease. It has been known that baicalein induces the formation of α-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 β-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 α-synuclein fibrillation. These baicalein-stabilized oligomers, added to the solution of aggregating α-synuclein, were able to noticeably inhibit its fibrillation. After prolonged coincubation, short fibrils were formed, suggesting an effective interaction of oligomers with monomeric α-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 α-synuclein oligomers might offer a potential strategy for the development of novel Parkinson's disease therapies.