Elucidating the aggregation number of dopamine-induced α-synuclein oligomeric assemblies.
Conventional methods to determine the aggregation number, that is, the number of monomers per oligomer, struggle to yield reliable results for large protein aggregates, such as amyloid oligomers. We have previously demonstrated the use of a combination of single-molecule photobleaching and substoichiometric fluorescent labeling to determine the aggregation number of oligomers of human α-synuclein, implicated in Parkinson's disease. We show here that this approach is capable of accurately resolving mixtures of multiple distinct molecular species present in the same sample of dopamine-induced α-synuclein oligomers, and that we can determine the respective aggregation numbers of each species from a single histogram of bleaching steps. We found two distinct species with aggregation numbers of 15-19 monomers and 34-38 monomers. These results show that this single-molecule approach allows for the systematic study of the aggregation numbers of complex supramolecular assemblies formed under different aggregation conditions.
Nanobiophysics, MESA+ Institute for Nanotechnology, University of Twente, Enschede, The Netherlands.
Niels
N
Zijlstra
Nanobiophysics, MESA+ Institute for Nanotechnology, University of Twente, Enschede, The Netherlands.
Mireille M A E
MM
Claessens
Nanobiophysics, MESA+ Institute for Nanotechnology, University of Twente, Enschede, The Netherlands.
Christian
C
Blum
Nanobiophysics, MESA+ Institute for Nanotechnology, University of Twente, Enschede, The Netherlands; Nanobiophysics, MIRA Institute for Biomedical Technology and Technical Medicine, University of Twente, Enschede, The Netherlands. Electronic address: subramaniam@amolf.nl.
Vinod
V
Subramaniam
D004298:Dopamine / Q000494:pharmacology*; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D038761:Photobleaching; D055503:Protein Multimerization / Q000187:drug effects*; D020836:Protein Structure, Quaternary; D051844:alpha-Synuclein / Q000737:chemistry*
D005456:Fluorescent Dyes; C497604:SNCA protein, human; D051844:alpha-Synuclein; D004298:Dopamine