Probing ensemble polymorphism and single aggregate structural heterogeneity in insulin amyloid self-assembly

Area of Science:

• Biophysics
• Materials Science
• Biochemistry

Background:

• Protein aggregates exhibit heterogeneity at nano- and micro-scales.
• This diversity influences biological systems, including neurodegenerative diseases and drug product immunogenicity.
• Analyzing diverse amyloid structures within ensembles is experimentally challenging.

Purpose of the Study:

• To investigate the structural heterogeneity of insulin protein aggregates.
• To develop a method for analyzing diverse amyloid structures at the single-particle level.
• To correlate molecular structure with aggregate morphology.

Main Methods:

• Fourier Transform Infrared Microscopy (micro-FTIR)
• Fluorescence Lifetime Imaging Microscopy (FLIM)
• Combined micro-FTIR and FLIM for submicron-scale analysis

Main Results:

• Demonstrated variable beta-structure architecture and content within insulin aggregates.
• Identified differences in beta-structure between spherulites and fibrils, and within individual spherulites.
• Revealed enhanced H-bond coupling in surface beta-structures of spherulites compared to their core.

Conclusions:

• The study successfully probes ensemble and single-particle heterogeneity in amyloid samples.
• Developed a scalable screening methodology for characterizing self-assembled protein structures.
• Findings are translatable to material sciences, drug quality control, and clinical imaging.