Identification of a helical intermediate in trifluoroethanol-induced alpha-synuclein aggregation

Because oligomers and aggregates of the protein alpha-synuclein (alpha S) are implicated in the initiation and progression of Parkinson's disease, investigation of various alpha S aggregation pathways and intermediates aims to clarify the etiology of this common neurodegenerative disorder. Here, we report the formation of short, flexible, beta-sheet-rich fibrillar species by incubation of alpha S in the presence of intermediate (10-20% v/v) concentrations of 2,2,2-trifluoroethanol (TFE). We find that efficient production of these TFE fibrils is strongly correlated with the TFE-induced formation of a monomeric, partly helical intermediate conformation of alpha S, which exists in equilibrium with the natively disordered state at low [TFE] and with a highly alpha-helical conformation at high [TFE]. This partially helical intermediate is on-pathway to the TFE-induced formation of both the highly helical monomeric conformation and the fibrillar species. TFE-induced conformational changes in the monomer protein are similar for wild-type alpha S and the C-terminal truncation mutant alpha S1-102, indicating that TFE-induced structural transitions involve the N terminus of the protein. Moreover, the secondary structural transitions of three Parkinson's disease-associated mutants, A30P, A53T, and E46K, are nearly identical to wild-type alpha S, but oligomerization rates differ substantially among the mutants. Our results add to a growing body of evidence indicating the involvement of helical intermediates in protein aggregation processes. Given that alpha S is known to populate both highly and partially helical states upon association with membranes, these TFE-induced conformations imply relevant pathways for membrane-induced alpha S aggregation both in vitro and in vivo.