Methods for characterizing the individual filament structures of amyloid peptide assemblies using atomic force microscopy.

A wide range of distinctive helical filamentous amyloid structures self-assembled from monomeric peptide or protein building blocks are found both in nature and in human disease states. Amyloid nano-fibrils are also being developed and synthetically made as novel peptide-based nanomaterials. In humans, accumulation of a range of amyloid structures, for example those formed from the amyloid-beta peptides in Alzheimer's disease, play a crucial role in the pathology of neurodegenerative and metabolic diseases. The diverse range of amyloid structures found is a manifestation of the amyloid structural polymorphism phenomenon. This is where different filament structures are assembled even from the same peptide or protein precursors. Due to the structural diversity of amyloid fibrils that can be found even in the same sample or the same disease state, an experimental method that allows structural analysis of individual amyloid filaments is required to understand the relationships between the polymorphic structures and the biological and physicochemical properties they elicit. Here, a method with a detailed protocol to analyze the structures of individual amyloid filament assemblies by topological Atomic Force Microscopy (AFM) imaging and Contact-Point Reconstruction AFM (CPR-AFM) image analysis is described. This approach to resolve the 3D shapes of amyloid polymorphs, one individual fibril at a time, allows mapping of the polymorphic landscapes of amyloid assemblies. It serves as an inexpensive, fast and effective experimental tool for individual filament level structural analysis, and offers new, exciting opportunities in elucidating population distributions of heterogeneous amyloid samples, rare amyloid structures within the populations, and structural variations between or within individual filaments. These are all key parts to experimental developments in therapeutic discovery and novel bio-nanomaterials applications.