Establishing the Composition of Alpha-Synuclein Oligomers using Single-Molecule Photobleaching

Abstract

Growing evidence suggests that oligomeric aggregation intermediates of the human alpha-synuclein protein are toxic to neurons in Parkinson's disease. Very little is known about the molecular details of these oligomeric aggregates. Conventional techniques to determine the number of monomers per oligomer, such as size exclusion chromatography or mass spectrometry, give estimates ranging from less than 10 to more than 60. It is unclear if this spread is due to influences from the technique used or if it reflects size heterogeneity of the oligomers.Single-molecule photobleaching allows for direct probing of the number of labeled monomers per oligomer, but is not typically applicable for large oligomers (>10 monomers). To overcome this problem, we develop a method that uses sub-stoichiometric labeling, that is, only a fraction of the monomers contain a fluorescent label, in combination with single-molecule photobleaching. The number of bleaching steps now gives the number of fluorescent labels instead of the number of monomers. Via the label probability mass function we link the number of fluorescent labels to the total number of monomers.Using this approach, we find a single, well defined alpha-synuclein oligomer consisting of 31 monomers. We also demonstrate that the aggregation is stochastic and that there is no influence of the fluorescent label on the aggregation. Fluorescence correlation spectroscopy data indicate a very loose packing of the oligomers. In combination with tryptophan fluorescence measurements, we propose a structural model for the oligomers.