Quantitative characterization of protein nanostructures using atomic force microscopy

The aggregation of proteins into fibrillar structures called amyloid is a characteristic of many diseases, including several neurodegenerative disorders. Although amyloid formation is inherent to several serious diseases, the mechanism of fibril formation and the modes of toxicity are not yet known. High concentrations of fibrillar aggregates of ¿-synuclein protein are found in the brains of patients suffering from Parkinson's disease. We exploit different contrast modes of high resolution atomic force microscopy (AFM) on fibrils formed by the wild-type alpha-synuclein protein, and by the familial disease-related A30P, E46K and A53T variants, to get more insight into the in vitro process of fibril assembly. From quantitative analysis of height images measured in tapping mode AFM, we obtained data that are compatible with a twisted hierarchical assembly model [1] for all protein variants. The E46K mutant displays the most distinct and smallest periodicity. The modulation depth for all mutants is very similar, and is smaller for wild-type protein commensurate with the lower fibril height. The detailed morphology observed in phase images indicates however that fibrils may also be formed through the association of fibril segments. To study the mechanical properties of fibrils we applied force while scanning in contact mode, resulting in characteristic deformation of protein fibrils with a periodicity corresponding to the modulation observed in tapping mode. Our observations suggest that the hierarchical assembly model may not be the exclusive mechanism of alpha-synuclein fibril assembly, but that multiple modes of fibril assembly play a role in alpha-synuclein fibril formation.