Studying the Effects of Anti-prion Compounds on PrP Folding at the Single-molecule Level

  • Author / Creator
    Rezajooei, Negar
  • Prion diseases, are associated with the misfolded form of the prion protein (PrPSc). The prion protein (PrP) has a unique means of transferring infectious diseases, based on a misfolded conformation. However, the mechanism of formation of PrPSc remains unclear owing to difficulties in defining the structure of PrPSc. Understanding the different folding pathways available to the protein and identifying those that lead to misfolding and aggregation, is possible through the use of single-molecule methods. However, finding these pathways is not easy due to the complexity in understanding the energy landscape. This thesis contains of two parts. In the first part, the sample preparation methods using different linking chemistries are explained. In the second part, the interaction between anti-prion ligands and PrP is investigated. In order to compare the effects of species on folding and misfolding pathways, we used single molecule force spectroscopy with the high resolution optical tweezers to study mouse PrP (MoPrP). Mouse PrP molecules suspended between two polystyrene beads via double stranded DNA handles. The optical tweezers were used to apply tension in order to denature a single molecule of MoPrP. As a result, the structural changes of a single molecule were monitored via end-to-end extension of the molecule. By moving the traps apart at a constant rate, the extension of the molecule increases monotonically until the protein unfolded causing a sudden increase in extension and concomitant drop in force. After analyzing the data, the results showed shorted contour length change matched the distance between one end of the molecule and either middle Cys(78) or Cys(113). We also confirmed internal labeling using an experiments which was performed with a fluorescent dye. In this experiment we used MoPrP without any external Cysteine (Cys). The result showed that internal Cys of MoPrP got labeled with the florescent dye. The similar results were observed by repeated the ,measurements on sample that was prepared using another attachment chemistry called click chemistry. The internal Cys labeling problem was fixed by replacing the reducing agent in our sample preparation method with a weaker reducing agent. Anti-prion ligands may offer a solution to prion diseases. Certain ligands that can interact with PrP, could potentially be considered as pharmacological chaperones. We also studied about the role of iron-tetrapyrrole, a potential pharmacological chaperones, on the folding pathways of Syrian hamster PrP (ShPrP). The Result suggested that Iron(III) meso-tetra (N-methyl04-pyridyl-prophine) (Fe-TMPyP) can bind to a single PrP molecule in two ways; either binding to the folded PrP monomer or to the unfolded PrP monomer. Fe-TMPyP can stabilize the native structure of single PrP molecules thermodynamically, kinetically, and mechanically by binding to the folded PrP. In addition, binding of Fe-TMPyP to the partially folded protein prevents the folding of Protein to the native state. Iron-tetrapyrrle, PrP-dimer always misfolded. In the presence of tetrapyrrole, the dimer sometimes shows folded states. We also planned to measure Syrian PrP (ShPrP) with another anti-prion compound called pentosan polysulphate (PPS). To do measurements we first needed to know the thermodynamics of interactions between protein and PPS. Measuring heat using isothermal titration calorimetry (ITC), is a way of finding this information before starting force extension measurements. We got a result of affinity binding (Kd value) between Kd ~ 0.05 uM to Kd ~ 5uM, consistent with the surface plasmon resonance (SPR) data given the large error. I want to do force extension measurements on ShPrP. In conclusion, single molecule methods give us opportunity to understand disease and biological function related to misfolded prion protein. The information derived from single molecule studies can be used to develop potential drugs which have anti-transmissible spongiform encephalopathies effects, such as sulphonated dyes, sulphated glycans, cyclic tetrapyrroles, quanacrine, and so on.

  • Subjects / Keywords
  • Graduation date
    Spring 2017
  • Type of Item
  • Degree
    Master of Science
  • DOI
  • 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
  • Institution
    University of Alberta
  • Degree level
  • Department
  • Supervisor / co-supervisor and their department(s)
  • Examining committee members and their departments
    • LeBlanc, Lindsay (Physics)
    • Freeman, Mark (Physics)
    • Tuszynski, Jack (Physics)