title: The effect of fluorescent labeling on α-synuclein fibril morphology.
abstract: The misfolding and aggregation of a small, natively unfolded protein α-synuclein (α-syn) is presumably an important factor in the development of Parkinson's disease. However, the mechanism of α-syn aggregation into amyloid fibrils and their morphology are not well understood. To elucidate the aggregation kinetics and the morphology of aggregates by the use of fluorescent techniques the protein needs to be suitably labeled. In this study, using atomic force microscopy, we demonstrate a significant effect of fluorescent labels on the α-syn fibrillization process. We studied in detail the morphology of α-syn aggregates as a function of the composition of mixtures of labeled and wild type (WT) α-syn in solution using different types of fluorescent dyes. Although the overall charge of the fluorophores we used and their chemical structure varied significantly, the morphology of α-syn fibrils changed in a similar way in all cases. The increase in the fraction of labeled α-syn in solution led to shortening of the fibrils as compared to those from WT-only α-syn, whereas the height of the fibrils remained mainly unaffected. The twisted fibril morphology observed in the WT and A140C α-syn mutant completely disappeared when the A140C α-syn mutant was 100% fluorescently labeled.
authors.affiliation: Leiden Institute of Physics, Leiden University, Leiden, The Netherlands.
authors.forename: M
authors.initials: M
authors.lastname: Mučibabić
authors.affiliation: Leiden Institute of Physics, Leiden University, Leiden, The Netherlands.
authors.forename: M M
authors.initials: MM
authors.lastname: Apetri
authors.affiliation: Leiden Institute of Physics, Leiden University, Leiden, The Netherlands.
authors.forename: G W
authors.initials: GW
authors.lastname: Canters
authors.affiliation: Leiden Institute of Physics, Leiden University, Leiden, The Netherlands. Electronic address: aartsma@physics.leidenuniv.nl.
authors.forename: T J
authors.initials: TJ
authors.lastname: Aartsma
mesh_terms: D000682:Amyloid / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D007700:Kinetics; D018625:Microscopy, Atomic Force / Q000379:methods; D010300:Parkinson Disease / Q000378:metabolism; D066263:Protein Aggregation, Pathological / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods; D051844:alpha-Synuclein / Q000378:metabolism*
chemical_list: D000682:Amyloid; D005456:Fluorescent Dyes; D051844:alpha-Synuclein
keywords: Atomic force microscopy; Fibrillization; Fluorescent labeling; Morphology; α-Synuclein