Amyloid fibrils and amorphous aggregates are two types of aberrant aggregates associated with protein misfolding diseases. Although they differ in morphology, the two forms are often treated indiscriminately. β 2 -microglobulin (β2m), a protein responsible for dialysis-related amyloidosis, forms amyloid fibrils or amorphous aggregates depending on the NaCl concentration at pH 2.5. We compared the kinetics of their formation, which was monitored by measuring thioflavin T fluorescence, light scattering, and 8-anilino-1-naphthalenesulfonate fluorescence. Thioflavin T fluorescence specifically monitors amyloid fibrillation, whereas light scattering and 8-anilino-1-naphthalenesulfonate fluorescence monitor both amyloid fibrillation and amorphous aggregation. The amyloid fibrils formed via a nucleation-dependent mechanism in a supersaturated solution, analogous to crystallization. The lag phase of fibrillation was reduced upon agitation with stirring or ultrasonic irradiation, and disappeared by seeding with preformed fibrils. In contrast, the glass-like amorphous aggregates formed rapidly without a lag phase. Neither agitation nor seeding accelerated the amorphous aggregation. Thus, by monitoring the kinetics, we can distinguish between crystal-like amyloid fibrils and glass-like amorphous aggregates. Solubility and supersaturation will be key factors for further understanding the aberrant aggregation of proteins.protein aggregation | metastability | glass transition | ultrasonication

Background: Protein oligomers are implicated as cytotoxic membrane-disrupting agents in neurodegenerative diseases. Results: The small molecule EGCG, which inhibits ␣-synuclein oligomer toxicity, moderately reduces membrane binding and immobilizing the oligomer C-terminal tail. Conclusion:The ␣-synuclein oligomer may disrupt membranes by vesicle destabilization rather than pore formation. Significance: Limited reduction of oligomer membrane affinity may be sufficient to prevent cytotoxicity.

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 ␤2-microglobulin (␤2-m), a protein responsible for dialysisrelated amyloidosis, we present that ultrasonication pulses are useful for preparing monodispersed amyloid fibrils of minimal size with an average molecular weight of Ϸ1,660,000 (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.␤2-microglobulin ͉ dialysis-related amyloidosis ͉ protein misfolding ͉ analytical ultracentrifugation A myloid fibrils are supramolecular assemblies exhibiting a long unbranched fibrillar morphology Ϸ10 nanometers in diameter, the deposition of which is associated with Ͼ30 degenerative diseases including Alzheimer's disease, prion disease, and dialysis-related amyloidosis (1-3). The past decade has seen progress in our biophysical understanding of amyloid fibrils using various approaches including solution and solid state NMR (4-6) and X-ray crystallography (7,8). However, the polymeric properties of amyloid fibrils, where huge size and heterogeneous nature result in insoluble and noncrystalline assemblies, are an obstacle to techniques such as X-ray crystallography, solution NMR spectroscopy and other conventional spectroscopic measurements. To overcome the analytical problems confronting studies of amyloid fibrils, it is worth establishing a strategy for producing monodispersed samples of amyloid fibrils. If amyloid fibrils of a well-defined molecular weight and improved solubility are formed reproducibly, a more general application of various types of fibril samples to a series of preexisting spectroscopic measurements will be accomplished.As a strategy to produce amyloid fibrils of uniform and minimal size, ultrasonication has several potential applications. Although ultrasonication was originally used to prepare seeds from preformed fibrils (9), which were further applied to the amplification of infectious prion proteins (10, 11), ultrasonication-dependent fragmentation is becoming an important approach to analyzing the properties of fibrils (12, 13). However, its strong effect of agitation has recently been found to accelerate the fibril nucleation of several proteins and peptides (14-18). Interestingly, amyloid fibrils produced by ultrasonicationinduced fibrillation were very short with apparently similar lengths as determined by AFM (15,19), suggesting that short fibrils of homogeneous molecular size are formed efficiently under ultrasonication in combination with the...

Although alcohols are useful cosolvents for producing amyloid fibrils, the underlying mechanism of alcohol-dependent fibrillation is unclear. We studied the alcohol-induced fibrillation of hen egg-white lysozyme at various concentrations of ethanol, 2,2,2-trifluoroethanol (TFE), and 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). Under the conditions where the alcohol-denatured lysozyme retained metastability, ultrasonication effectively triggered fibrillation. The optimal alcohol concentration depended on the alcohol species. HFIP showed a sharp maximum at 12-16%. For TFE, a broad maximum at 40-80% was observed. Ethanol exhibited only an increase in fibrillation above 60%. These profiles were opposite to the equilibrium solubility of lysozyme in water/alcohol mixtures. The results indicate that although fibrillation is determined by solubility, supersaturation prevents conformational transitions and ultrasonication is highly effective in minimizing an effect of supersaturation. We propose an alcohol-dependent protein misfolding funnel useful for examining amyloidogenicity. This misfolding funnel will apply to fibrillation under physiological conditions where biological environments play important roles in decreasing the solubility.