Characterisation of polymorphic amyloid fibrils associated with disease and biological function by atomic force microscopy

The remarkable ability of some proteins with a single sequence to fold and assemble into b-sheet rich structures in diverse ways, a phenomenon often referred to as amyloid polymorphism, is a challenging and unresolved aspect of protein folding and assembly. Structural studies have brought insights into the structural differences in amyloid fibrils assembly and morphology, using cutting-edge imaging techniques. Although, structural polymorphism has been established for different amyloid fibrils, however, the structural polymorphism of individual amyloid fibril and the effect of polymorphism on their intrinsic stability remained unclear. Here, we characterised the degree of structural heterogeneity in α-synuclein wild type (WT) and two of its familial point mutations (A30P and A53T). We also evaluated the intrinsic stability of these morphologically different variants of α-synuclein amyloid fibril toward fragmentation. To further bring insights into our understanding about different types of amyloid fibrils, we explored polymorphism in well-studied yeast functional amyloid fibril, Sup35NM, and observed the impact of environmental factors on amyloid fibril assembly, morphology, and intrinsic stability toward fragmentation. Here, we used atomic force microscopy (AFM) to study the morphological properties of amyloid fibrils, and employed the recent advancement in AFM image analysis to quantitatively assess amyloid fibril polymorphism. Thioflavin T kinetic assay was also used to probe amyloid fibril assembly. Fibril polymorphism was observed across the different variants of α synuclein amyloid fibrils and that mutation in precursor protein can substantially affect the rate of kinetic as well as structural diversity. The data showed that amyloid formed in vitro from these A30P and A53T α-synuclein variants is more resistant towards 2 fragmentation. Furthermore, we presented structural polymorphism of Sup35NM amyloid fibril polymorphism and the activation of heat shock 104 (Hsp104) in cell extract may inhibit fibrilization and lead to formation of morphologically distinct amyloid fibril, in comparison to the fibrils assembled in buffer or cell extract, before treated with heat shock the effect of (Hsp104). In summary, this work demonstrates how physiological and environmental factors affect different amyloid fibrils morphology and their stability.