Albany 2013: Book of Abstracts
June 11-15 2013
©Adenine Press (2012)
Getting A Grip On Alpha-Synuclein Amyloid Oligomers
Misfolding and aggregation of proteins into nanometer-scale fibrillar assemblies is a hallmark of many neurodegenerative diseases. Aggregation of the human alpha-synuclein protein is implicated in the etiology of Parkinson’s disease. A particularly relevant question is the role of early oligomeric aggregates of alpha-synuclein in modulating the dynamics of protein aggregation, and in the interactions with essential cellular components. However, very little is known about the molecular details of these aggregate species. For large protein aggregates, such as alpha-synuclein oligomers, it is very difficult to determine the number of monomers that form an oligomer using conventional techniques. We have developed 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 to determine the number of monomers per oligomer (Zijlstra et al., 2012). The number of bleaching steps gives the number of fluorescent labels per oligomer. Knowing the exact label density, that is, the fraction of labeled monomers at the start of the aggregation, we can correlate the number of fluorescent labels per oligomer to the total number of monomers. Using this method, we can determine the composition, probe the distribution in the number of monomers per oligomer, and investigate the influence of the fluorescent label on the aggregation process. For wild-type alpha-synuclein, we find no distribution in the number of monomers per oligomer and find a single, well-defined oligomeric species consisting of ~30 monomers per oligomer. On the other hand, for oligomers formed in the presence of dopamine, we find a distinctly bimodal distribution, suggesting the existence of two populations of oligomeric species.
This work was financially supported by the “Nederlandse Organisatie voor Wetenschappelijk Onderzoek” (NWO) through the NWO-CW TOP program number 700.58.302. We further acknowledge support from the “Stichting Internationaal Parkinson Fonds.”
Nanobiophysics, MIRA Institute, University of Twente
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