Early events in insulin fibrillization studied by time-lapse atomic force microscopy

Area of Science:

• Biochemistry
• Biophysics
• Materials Science

Background:

• Protein aggregation into amyloid fibrils has significant medical implications and reveals fundamental self-organization principles.
• A wide range of proteins can form similar fibril structures under specific conditions, indicating a general mechanism for polypeptide chains.

Purpose of the Study:

• To investigate the initial events preceding amyloid fibril formation in zinc-free human insulin solutions.
• To elucidate the kinetic mechanisms governing the early stages of protein aggregation.

Main Methods:

• Utilizing time-lapse atomic force microscopy to observe protein aggregation dynamics.
• Incubating zinc-free human insulin solutions at low pH and high temperature.

Main Results:

• A steady-state distribution of protein oligomers with a quasiexponential tail forms within minutes of heating.
• This metastable prefibrillar phase persists for hours before observable fibrillar aggregates emerge.
• The prefibrillar phase kinetics are primarily governed by a coagulation-evaporation mechanism, with concentration as a critical factor.

Conclusions:

• The early stages of insulin amyloid fibril formation are characterized by a metastable oligomeric phase.
• A simple coagulation-evaporation kinetic mechanism, influenced by concentration, controls this prefibrillar phase.
• Thermal concentration fluctuations likely play a crucial role in the nucleation of amyloid fibrils.