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Aggregation of α-synuclein monomers and engineered oligomers in solution

  • Author / Creator
    Li, Xi
  • α-Synuclein is a protein that has been found as fibrillar aggregates in Lewy bodies in the brain of Parkinson’s disease patients. Though the cause of the Parkinson’s disease is unknown, previous research suggest that there is a close association between the disease and the toxicity of the intermediary α-synuclein oligomers in human neurons. Therefore, it is important to investigate the aggregation behaviour of intermediary oligomers. To do this we investigate aggregation of protein constructs that are monomers (Snca1), or engineered dimers (Snca2), tetramers (Snca4), and octamers (Snca8) of α-synuclein with a C terminal cysteine. Single-molecule fluorescence methods were used to study the molecular interactions between pairs of these oligomers under “physiological” aggregation conditions (10 mM PBS at pH = 7.4 at 37 °C). To be specific, we applied dual-color fluorescence cross correlation spectroscopy (dual-color FCCS) to study the self-aggregation (aggregation of one kind of α-synuclein) and the cross-aggregation (aggregation between the monomeric and oligomeric α-synuclein) of α-synuclein. The engineered α-synuclein monomers, dimers, tetramers and octamers were labelled with either Oregon Green 488 maleimide (green dye) or Cy5-tetrazine (red dye). A green dye labelled protein was incubated with a red dye labelled protein at micromolar concentrations with continuous shaking at 250 rpm and diluted aliquots of the aggregation mixture was measured by dual-color FCCS at nanomolar concentrations after different incubation times. The experimental results indicate that the engineered α-synuclein octamers aggregate faster and to a greater extent than monomers and dimers, with the tetramers being intermediate. The engineered oligomers preferred to incorporate themselves rather than the monomer into aggregates. Therefore, these oligomer constructs do not appear to seed the aggregation of monomers.

  • Subjects / Keywords
  • Graduation date
    2015-11
  • Type of Item
    Thesis
  • Degree
    Master of Science
  • DOI
    https://doi.org/10.7939/R38W38C8J
  • 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
    Master's
  • Department
    • Department of Chemistry
  • Supervisor / co-supervisor and their department(s)
    • Petersen, Nils (Department of Chemistry)
  • Examining committee members and their departments
    • Lucy, Charles (Department of Chemistry)
    • Serpe, Michael (Department of Chemistry)