Understanding the mechanism of amyloid fibril formation, a nucleation-dependent process influenced by physicochemical conditions, is crucial for eradicating amyloidosis, as it represents the initial step in the pathological cascade of the disease. In this study, we develop a miniaturized ultrasonic platform for the amyloid fibril study, μHANABI, which enables reproducible and rapid analysis of amyloid formation kinetics. Leveraging the robustness of the developed system, we investigate the nucleation rate of α-synuclein, an intrinsically disordered protein, and β2-microglobulin, a natively folded protein, at various temperatures and salt concentrations. The lag time for nucleation exhibits a clear dependence on salt concentration: α-synuclein amyloid formation is monotonically accelerated with increasing salt concentration, whereas β2-microglobulin exhibits a U-shape dependence. This contrast can be rationalized by evaluating the degree of supersaturation as a function of temperature and salt concentration, linking the solubility-limited phase transition with the folding-related two-state transition. These results demonstrate the superior performance of μHANABI as an effective platform for physicochemical studies of amyloid formation mechanisms, and the obtained results provide a physicochemical understanding of amyloid nucleation coupled with the folding reaction.
Ota et al. (Thu,) studied this question.