A robust yet simple method to generate fluorescent amyloid nanofibers

Area of Science:

• Biochemistry
• Materials Science
• Molecular Biology

Background:

• Covalent fluorophore tagging is crucial for studying biological processes like protein aggregation and interactions.
• Existing methods for creating fluorescently tagged proteins are often expensive, complex, and can alter protein structure.
• Understanding amyloidogenesis, cellular internalization, and crosstalk requires effective tools for monitoring these processes.

Purpose of the Study:

• To develop a simple, robust, and affordable method for fabricating fluorescent amyloid nanofibers.
• To create fluorescent amyloid nanofibers that retain native aggregation kinetics and β-sheet conformers.
• To enable monitoring of cellular internalization and Förster Resonance Energy Transfer (FRET) signals in amyloid structures.

Main Methods:

• A coassembly-reaction route was employed to synthesize fluorescent amyloid nanofibers.
• The method was validated using insulin, lysozyme, amyloid-beta 1-42 (Aβ_1-42), and metabolites.
• Stability of incorporated fluorophores was assessed through serial dilutions and prolonged storage.

Main Results:

• Successfully fabricated fluorescent amyloid nanofibers from various proteins and metabolites.
• The coassembly method preserved the aggregation kinetics and β-sheet conformation of the nanofibers.
• Incorporated fluorophores demonstrated excellent stability, resisting leaching under various conditions.
• The fluorescent nanofibers facilitated monitoring of cellular uptake and FRET signals.

Conclusions:

• The developed coassembly-reaction route provides a simple and affordable method for creating stable fluorescent amyloid nanofibers.
• This technique overcomes limitations of traditional covalent tagging, offering a valuable tool for amyloid research in vitro and in vivo.
• The fluorescent nanofibers enable advanced studies on amyloidogenesis, cellular interactions, and fibril dynamics.