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Permanent link (DOI): https://doi.org/10.7939/R38W38C8J

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Aggregation of α-synuclein monomers and engineered oligomers in solution Open Access

Descriptions

Other title
Subject/Keyword
α-synuclein
Fluorescence correlation spectroscopy (FCS)
Dual-color fluorescence cross-correlation spectroscopy (dual-color FCCS)
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Li, Xi
Supervisor and department
Petersen, Nils (Department of Chemistry)
Examining committee member and department
Serpe, Michael (Department of Chemistry)
Lucy, Charles (Department of Chemistry)
Department
Department of Chemistry
Specialization

Date accepted
2015-05-05T09:22:57Z
Graduation date
2015-11
Degree
Master of Science
Degree level
Master's
Abstract
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.
Language
English
DOI
doi:10.7939/R38W38C8J
Rights
This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for the purpose of private, scholarly or scientific research. 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.
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