Single Molecule AFM Force Spectroscopy Analysis of Alpha-Synuclein Misfolding

Abstract

Protein misfolding is a transient state during self-assembly into aggregates defining the molecular mechanism of the development of Alzheimer's, Parkinson's and other neurodegenerative diseases. Misfolding and aggregation of alpha-synuclein (α-Syn) is tightly linked to the development of Parkinson's disease. Here we applied single molecule AFM force spectroscopy (SMFS) to probe transient misfolded states of α-Syn measuring pair-wise interactions between individual α-Syn molecules at conditions that induce conformational transitions associated with enhanced aggregation. In the SMFS approach we probed the interactions between α-Syn covalently attached to the AFM probe and substrate by the C-terminal cysteine. We show that at conditions close to physiological, addition of spermidine results in dramatic increase of the protein's propensity to misfold. Additionally, using SMFS we detected and characterized misfolded dimers of α-Syn, the simplest aggregated form of α-Syn. Our results demonstrate that more than one segment within the protein molecule is responsible for the initial association of α-Syn into dimers and potentially into higher-order oligomers and fibrils. This finding suggests that even the first step of α-Syn self-assembly (dimerization) possesses a certain degree of heterogeneity. We hypothesize that these different misfolded conformations can lead to different types of oligomers and define the aggregation pathway. The marked differences in the misfolding patterns between WT α-Syn and single point mutants might be responsible for the higher propensity of the mutants to aggregate and cause early-onset PD.The work was supported by grants to YLL from National Institutes of Health (1P01GM091743-01A1 and 1 R01 GM096039-01A1), U.S. Department of Energy Grant DE-FG02-08ER64579, National Science Foundation (EPS - 1004094) and the Nebraska Research Initiative.