Distinguishing crystal-like amyloid fibrils and glass-like amorphous aggregates from their kinetics of formation

Abstract

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

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

“…10(a)]. 51) Yoshimura et al 51) also proposed a general phase diagram for various conformational states of peptides and proteins determined by conformational uniqueness [ Fig. 10(b)].…”

“…Yoshimura et al 51) monitored the effects of ultrasonication on fibrillation of 2-m in real time. The sample solution in a cuvette was irradiated with ultrasonication pulses by tightly attaching an ultrasonic generator to the sidewall of the cuvette as shown in Fig.…”

“…When the effects of ultrasonic pulses or seeding were examined, the solutions were stirred. The figures were reproduced from Yoshimura et al 51) On the basis of the results, a phase diagram was constructed, representing amyloid formation and amorphous aggregation of 2-m [ Fig. 10(a)].…”

Supersaturation-limited Amyloid Fibrillation of Insulin Revealed by Ultrasonication

“…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).…”

“…Under labile conditions in which the driving forces of precipitation increase, spontaneous crystallization can occur after a certain period of lag time. Further increases in driving forces have been shown to result in the rapid breakdown of supersaturation, leading to the formation of glassy amorphous aggregates (19). The importance of supersaturation has recently been reported in the context of widespread aggregation and neurodegenerative diseases (24).…”