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Free keywords:
α-synuclein; atomic force microscopy; β-sheet; soluble oligomer; amyloid fibril.
Abstract:
Self-assembly of α−synuclein resulting in protein aggregates of diverse morphology has been implicated in the pathogenesis of Parkinson’s disease and other neurodegenerative disorders known as synucleinopathies. Apart from its biomedical relevance, this aggregation process is representative of the interconversion of an unfolded protein into nanostructures with typical amyloid features. We have used in situ tapping mode atomic force microscopy (AFM) to continuously monitor the self-assembly of wild-type α-synuclein, its disease-related mutants A30P and A53T, and the C-terminally truncated variant α-synuclein(1-108). Different aggregation modes were observed depending on experimental conditions, i.e. pH, protein concentration, polyamine concentration, temperature and the supporting substrate. At pH 7.5, in the absence of the biogenic polyamines spermidine or spermine, elongated sheets 1.1(±0.2)nm in height and presumably representing individual β-sheet structures, were formed on mica substrates within a few minutes. Their orientation was directed by the crystalline substructure of the substrate. In contrast, sheet formation was not observed with hydrophobic highly oriented pyrolytic graphite (HOPG) substrates, suggesting that negatively charged surfaces promote α-synuclein self-assembly. In the presence of spermidine or spermine 5.9(±1.0) nm high spheroidal structures were preferentially formed, sharing characteristics with similar structures previously reported for several amyloidogenic proteins and linked to neurotoxicity. Alpha-synuclein spheroid formation depended critically on polyamine binding to the Cterminus, revealing a promoting effect of the C-terminus on α-synuclein assembly in the bound state. In rare cases, fibril growth from spheroids or preformed aggregates was observed. At pH 5.0, fibrils were formed initially and incorporated into amorphous aggregates in the course of the aggregation process, providing evidence for the potential of amyloid fibril surfaces to act as nucleation sites in amorphous aggregation. This study provides a direct insight into different modes of α-synuclein self-assembly and identifies key factors modulating the aggregation process.
