High-throughput Analysis of Ultrasonication-forced Amyloid Fibrillation Reveals the Mechanism Underlying the Large Fluctuation in the Lag Time

Abstract

Background: Ultrasonication effectively breaks supersaturation and forces amyloid fibrillation. Results: A high-throughput analysis of amyloid fibrillation showed that, although the lag time varied depending on the conditions, its coefficient of variation was constant. Conclusion: The large fluctuation in the lag time originates from a process associated with a common amyloidogenic intermediate. Significance: High-throughput analysis is powerful enough to clarify the mechanisms of supersaturation-limited phase transitions of proteins.

Supersaturation-limited and Unlimited Phase Transitions Compete to Produce the Pathway Complexity in Amyloid Fibrillation

“…These ultrasonic power-dependent decreases in the lag time and ThT fluorescence (Fig. 2C) were consistent with our previous studies on ␤2m (22,25) and findings on ␣-synuclein fibrils (27) and amyloid ␤-(1-40) fibrils (26), implying that extensive ultrasonication transformed the preformed fibrils to amorphous aggregates. In addition, the decrease observed in maximal ThT fluorescence with an increase in ultrasonic power suggested the direct formation of amorphous aggregates as well as amyloid fibrils.…”

“…The decrease in ThT fluorescence was accelerated when the lag time was shorter, suggesting that this reduction was caused by the ultrasonication-dependent transformation of preformed fibrils into distinct conformational states (22,25,26). Combined with the salt concentration-dependent competition between fibrillation and amorphous aggregation, we showed that a competitive mechanism, in which various amyloidogenic and non-amyloidogenic aggregates competed (Fig.…”

“…Measurements were performed at 25°C using a quartz cuvette with a 1-mm path length, and the results were expressed as mean residue ellipticity []. AFM images were obtained using a Digital Instruments Nanoscope IIIa scanning microscope (Veeco Instruments Inc., Plainview, NY) as described previously (25). Regarding TEM measurements, 10-fold diluted samples were spotted onto a collodion-coated copper grid (Nisshin EM Co., Tokyo, Japan).…”

“…When we monitored the ultrasonication-dependent fibrillation of proteins by thioflavin T (ThT), we often observed a significant decrease in ThT fluorescence after the burst-phase increase (22,(25)(26)(27). The decrease in ThT fluorescence was accelerated when the lag time was shorter, suggesting that this reduction was caused by the ultrasonication-dependent transformation of preformed fibrils into distinct conformational states (22,25,26).…”

“…In the case of ultrasonication-forced fibrillation, we suggested that interactions with the hydrophobic surfaces of cavitation bubbles condensed proteins, leading to the breakdown of supersaturation and then fibrillation (8,23). Ultrasonication is now recognized as one of the important approaches for elucidating the mechanisms underlying amyloid fibrillation (23)(24)(25).…”