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Single-molecule force spectroscopy of alpha-synuclein

  • Author / Creator
    Solanki, Allison K
  • Intrinsically disordered proteins (IDPs) represent a large subpopulation of the proteome, and are characterized by high structural plasticity and a predisposition to aggregate. These aggregates can accumulate inside or outside the cell, often forming amyloid plaques that are implicated in a variety of maladies, including Alzheimer’s disease, type II diabetes and Parkinson’s disease. Despite many advances in elucidating aggregation mechanisms, much is still unknown about the early oligomeric states, which are the suspected toxic agents in disease, since their transient nature makes them hard to study with traditional experimental methods. Here we have investigated the initial stages of aggregation of one such IDP, α-synuclein, a protein that is involved in Parkinson’s disease and other related dementias. To do so, we developed assays for characterizing the transient structure, stability, and kinetics of monomers and oligomers using optical tweezers with high spatial and temporal resolution. Measurements of the molecular extension as the proteins unfolded under tension revealed that even small oligomers could form numerous metastable structures, with a surprisingly broad range of sizes. Moreover, our data also revealed rapid fluctuations at low force, arising from the folding of two different classes of structure that were only marginally stable. The energy landscape for these transitions was characterized via the force-dependent kinetics derived from correlation analysis of the extension trajectories. The barriers were small, only a few kBT, but the diffusion was slow, revealing a landscape that is flat but rough. This thesis presents the first measurements of α-synuclein using optical tweezers, and also provides the first experimentally reconstructed energy landscape for an IDP.

  • Subjects / Keywords
  • Graduation date
    2014-11
  • Type of Item
    Thesis
  • Degree
    Doctor of Philosophy
  • DOI
    https://doi.org/10.7939/R3RJ4934Q
  • License
    This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.
  • Language
    English
  • Institution
    University of Alberta
  • Degree level
    Doctoral
  • Department
    • Department of Physics
  • Supervisor / co-supervisor and their department(s)
    • Woodside, Michael T (Physics)
  • Examining committee members and their departments
    • Plotkin, Steven (Physics and Astronomy, UBC)
    • Freeman, Mark (Physics)
    • Sim, Valerie (Medicine)
    • Tuszynski, Jack (Physics)