Quantitative Analysis of Amyloid Fibril Nucleation by Linking Folding and Nucleation Pathways Using a Robust...

Area of Science:

• Biochemistry
• Biophysics
• Materials Science

Background:

• Amyloid fibril formation is a nucleation-dependent process critical to amyloidosis.
• Physicochemical conditions significantly influence amyloidogenesis.
• Understanding nucleation mechanisms is key to developing therapeutic strategies.

Purpose of the Study:

• To develop a miniaturized ultrasonic platform (μHANABI) for rapid and reproducible amyloid formation kinetics analysis.
• To investigate the nucleation rates of α-synuclein and β2-microglobulin under varying physicochemical conditions.
• To elucidate the relationship between protein folding and amyloid nucleation.

Main Methods:

• Development of the miniaturized ultrasonic platform, μHANABI.
• Kinetic analysis of amyloid fibril formation for α-synuclein and β2-microglobulin.
• Systematic variation of temperature and salt concentration to study nucleation rates.

Main Results:

• The μHANABI platform enables reproducible and rapid analysis of amyloid formation kinetics.
• α-synuclein nucleation rate increases monotonically with salt concentration.
• β2-microglobulin nucleation rate shows a U-shaped dependence on salt concentration.
• Observed differences in nucleation kinetics are linked to supersaturation and protein folding transitions.

Conclusions:

• μHANABI is a high-performance platform for physicochemical studies of amyloid formation.
• The study provides a physicochemical understanding of amyloid nucleation coupled with protein folding.
• Distinct salt concentration dependencies highlight different nucleation mechanisms for intrinsically disordered and natively folded proteins.