Ultrasonication: An Efficient Agitation for Accelerating the Supersaturation-Limited Amyloid Fibrillation of Proteins

Abstract

Amyloid fibrils are self-assemblies of proteins with an ordered cross-β architecture. Because they are associated with serious disorders, understanding their structure and mechanism of fibrillation is important. Irradiation with ultrasonication leads to fragmentation of amyloid fibrils, useful for seeding experiments. Recently, ultrasonication has been found to trigger the spontaneous formation of fibrils in solutions of monomeric amyloidogenic proteins. The results indicate that amyloid fibrillation is similar to the crystallization of solutes from a supersaturated solution. The accelerating effects of ultrasonication on amyloid fibrillation suggest that cavitation microbubbles play a key role in effectively converting the metastable state of supersaturation to the labile state, leading to spontaneous fibrillation. Moreover, ultrasonic irradiation would be promising for a high-throughput screening assay of amyloid fibrillation, advancing the study of supersaturation-limited amyloidogenesis.

Supersaturation-limited Amyloid Fibrillation of Insulin Revealed by Ultrasonication

“…Previous studies showed that crystal-like ordered states including amyloid fibrils do not form rapidly because of the high free energy of nucleation (19,23), which leads to supersaturation in which, in the absence of agitations to induce nucleation, the solutes may retain apparent solubility indefinitely under the conditions of metastability. On the other hand, when solvent-excluded conformations are glassy amorphous aggregates, which is common for larger denatured proteins, aggregation has been shown to occur rapidly without a lag phase.…”

“…The same effects may be induced by other types of agitations such as stirring. One possible mechanism is the condensation of amyloidogenic proteins at the water/air interface produced by ultrasonication-induced cavitation or stirring (19,23). Local increases in the protein concentration at the water/air interface have been shown to lead to glassy aggregates, in which a template-competent conformation may emerge.…”

“…The importance of supersaturation has recently been reported in the context of widespread aggregation and neurodegenerative diseases (24). Although the physicochemical mechanism of supersaturation remains unclear, we have suggested that ultrasonication is one of the most powerful agitations breaking the otherwise persistent metastability of supersaturation (19,23,(25)(26)(27).…”

“…The residual solution was removed with compressed air and air dried for 10 s. AFM images were obtained using a Nano Scope IIIa (Digital Instruments), as reported previously (23,39).…”

Benzalkonium Chloride Accelerates the Formation of the Amyloid Fibrils of Corneal Dystrophy-associated Peptides

“…In the present study, ultrasonic treatment, one of the most powerful methods for accelerating spontaneous fibrillation in vitro, was used to efficiently investigate the amyloid fibrillation of keratoepithelin-derived peptides (27)(28)(29)(30). We used the wildtype R-peptide and disease-associated C-type and H-type peptides.…”