Single-Molecule Studies of Factors Protecting Against Prion Protein Misfolding
Creators: Patra, Shubhadeep
Publisher: name: University of Alberta Library
Year: 2024

<p>Proteins have generally evolved to fold properly into the particular native conformation to be able to execute specific biological functions. Sometimes, the protein deviates from the correct folding process which results in the formation of a non-native structure and misfolding of the protein occurring both inside and outside the cells. Misfolding of the prion proteins, PrP, is associated with a wide range of fatal neurodegenerative diseases called prion diseases. Several aspects of protection against PrP misfolding have been identified including small changes of only a few amino acids in the PrP sequences among species with differential prion disease susceptibilities, ligands that display anti-prion activities, and single-point mutations that confer resistance. However, the molecular mechanisms related to such protection remain elusive, partly because it is difficult to observe directly the rare and transient molecular events linked to the misfolding and characterize the complexity of the underlying energy landscape. Single-molecule methods have the potential to approach these difficulties and provide a powerful platform to study misfolding.<br>
In this thesis, we probed the folding of single PrP molecules featuring various factors that can protect against prion diseases using a high-resolution optical tweezer. We first compared the effects of sequence differences found in PrP from species with different degrees of prion disease-susceptibilities: disease-susceptible hamster PrP (HaPrP), highly disease-susceptible bank vole PrP (BvPrP), and disease-resistant dog PrP (CaPrP). We also investigated the effects on folding of two point-mutations, G127V and N159D, that can provide immunity against disease. Finally, we explored the effects of an anti-prion ligand, pentosan polysulfate (PPS), on the folding of PrP molecules.<br>
These experiments revealed detailed information about the folding behavior of the PrPs that include folding dynamics, intermediate states and structures, folding pathways, properties of energy barriers, stability of the structures, and an estimate of the energy landscape. We found that despite minor changes in sequences for PrP from species with different degrees of disease susceptibility, the unfolding and refolding behavior differed in several important aspects. Notably, we observed long-lived misfolded states in monomers of BvPrP, which is a universal prion disease receptor, deducing the presence of at least three structurally distinct misfolded states. Studies of the antiprion compound PPS revealed the likely importance of unfolded states of PrP in the mechanism for propagated misfolding: PPS stabilized partially unfolded intermediate states as well as the fully unfolded states of HaPrP and BvPrP, displaying heterogeneous interactions, and bound roughly 40-fold more tightly to unfolded states than to the native state. Finally, when we added the protective mutations G127V or N159D to BvPrP, we found that they abolished misfolding. The two mutations resulted in different folding behavior, but they both redirected the dominant folding pathways away from a particular partially folded intermediate into the same new intermediate states that were not present in the folding of the wild-type protein, suggesting that these states were protective against misfolding. By discovering how various protective factors alter the structural dynamics associated with PrP misfolding, we gained new insights into PrP misfolding and potential mechanisms conferring protection against misfolding.</p>