Secondary Structure of α-Synuclein Oligomers: Characterization by Raman and Atomic Force Microscopy

Formation of α-synuclein aggregates is proposed to be a crucial event in the pathogenesis of Parkinson's disease. Large soluble oligomeric species are observed as probable intermediates during fibril formation and these, or related aggregates, may constitute the toxic element that triggers neurodegeneration. Unfortunately, there is a paucity of information regarding the structure and composition of these oligomers. Here, the morphology and the conformational characteristics of the oligomers and filaments are investigated by a combined atomic force microscopy (AFM) and Raman microscopic approach on a common mica surface. AFM showed that in vitro early stage oligomers were globular with variable heights, while prolonged incubation caused the oligomers to become elongated as protofilaments. The height of the subsequently formed α-synuclein filaments was similar to that of the protofilaments. Analysis of the Raman amide I band profiles of the different α-synuclein oligomers establishes that the spheroidal oligomers contain a significant amount of α-helical secondary structure (47%), which decreases to about 37% in protofilaments. At the same time, when protofilaments form, β-sheet structure increases to about 54% from the ∼29% observed in spheroidal oligomers. Upon filament formation, the major conformation is β-sheet (66%), confirmed by narrowing of the amide I band and the profile maximum shifting to 1667 cm−1. The accumulation of spheroidal oligomers of increasing size but unchanged vibrational spectra during the fibrillization process suggests that a cooperative conformational change may contribute to the kinetic control of fibrillization.

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