Amyloid fibrils, crystal-like aggregates of denatured proteins, are associated with diseases such as dialysis-related amyloidosis (DRA)1. Preventing amyloid formation prior to clinical manifestation is important, and in vitro studies play a crucial role in understanding its onset. The formation occurs through a nucleation-growth mechanism2 from supersaturated solution of denatured monomers whose concentration exceeds its solubility3. However, due to a high energy barrier in nucleation, this process occurs after a long lag time. In the case of β2-microglobulin (β2m), associated with DRA, amyloid formation fails to occur easily under neutral pH because of the amyloid-resistant native structure. Therefore, the exact mechanism of DRA remains unclear. In this study, we investigated the mechanism of β2m amyloid formation by applying ultrasonication4 at elevated temperatures5, which significantly accelerates amyloid formation.
We first systematically investigate β2m amyloid formation at various NaCl concentrations between 50 °C and 80 °C with ultrasonic irradiation. For the ultrasonic experiment, we originally developed an instrument, which irradiates sample solutions in a micro-well plate with ultrasound, measuring the time course of amyloid formation. Amyloid formation was observed above 56°C, and it was significantly accelerated as the NaCl concentration increased above 60°C. Interestingly, in a 56-59 °C range, the formation slowed with increasing the NaCl concentration above about 700 mM, indicating a complex interaction between salt concentration and protein stability. In addition, thermal denaturation experiments further revealed that the denaturation midpoint at higher salt concentration is higher than those at lower ones, suggesting that the addition of salt essentially stabilizes the native monomer relative to denatured monomers. These findings indicate that two effects of salt on folding and amyloid formation are in competition, demonstrating the importance of understanding linkage between intramolecular folding and intermolecular amyloid formation to reveal the amyloid formation mechanism in vivo.