Abstract
α-Synuclein is a protein that aggregates as amyloid fibrils in the brains of patients with Parkinson’s disease and dementia with Lewy bodies. Small oligomers of α-synuclein are neurotoxic and are thought to be closely associated with disease. Whereas α-synuclein fibrillization and fibril morphologies have been studied extensively with various methods, the earliest stages of aggregation and the properties of oligomeric intermediates are less well understood because few methods are able to detect and characterize early-stage aggregates. We used fluorescence spectroscopy to investigate the early stages of aggregation by studying pairwise interactions between α-synuclein monomers, as well as between engineered tandem oligomers of various sizes (dimers, tetramers, and octamers). The hydrodynamic radii of these engineered α-synuclein species were first determined by fluorescence correlation spectroscopy and dynamic light scattering. The rate of pairwise aggregation between different species was then monitored using dual-color fluorescence cross-correlation spectroscopy, measuring the extent of association between species labelled with different dyes at various time points during the early aggregation process. The aggregation rate and extent increased with tandem oligomer size. Self-association of the tandem oligomers was found to be the preferred pathway to form larger aggregates: interactions between oligomers occurred faster and to a greater extent than interactions between oligomers and monomers, indicating that the oligomers were not as efficient in seeding further aggregation by addition of monomers. These results suggest that oligomer-oligomer interactions may play an important role in driving aggregation during its early stages.
Highlights
1000 Number of amino acids phase characterized by a very slow growth rate, during which monomers co-exist with transient small oligomers, followed by a dramatically increased growth rate of β-structured fibrillar material[33]
Single-molecule (SM) methods are well-suited to studying these early stages[45,46,47], and a variety of SM techniques have been used to study the interactions between α-synuclein molecules leading to aggregation, including fluorescence resonance energy transfer (FRET)[48,49,50,51,52], fluorescence correlation spectroscopy (FCS)[48,53,54], and force spectroscopy[55,56,57]
Measuring the extent of association between oligomeric species of different sizes at various points during the earliest stages of aggregation, we found that the tandem oligomers preferentially self-associated rather than acting as seeds to accelerate the addition of monomers
Summary
1000 Number of amino acids phase characterized by a very slow growth rate, during which monomers co-exist with transient small oligomers, followed by a dramatically increased growth rate of β-structured fibrillar material[33]. The heterogeneity of oligomers and their transitory nature[58] make it difficult to study their role in the α-synuclein aggregation process To address these challenges, we engineered tandem-repeat oligomers of α-synuclein having a specified size by connecting two, four, or eight monomers head-to-tail with a three-amino-acid linker between each repeated domain[55,56,59]. We engineered tandem-repeat oligomers of α-synuclein having a specified size by connecting two, four, or eight monomers head-to-tail with a three-amino-acid linker between each repeated domain[55,56,59] This approach, which has been previously applied to study aggregation in other proteins[60,61,62], allowed us to study the properties of oligomers of different sizes in a controlled way. Measuring the extent of association between oligomeric species of different sizes at various points during the earliest stages of aggregation, we found that the tandem oligomers preferentially self-associated rather than acting as seeds to accelerate the addition of monomers
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
