Single-molecule studies of prion protein folding and misfolding

• Author / Creator

  Yu, Hao

• Protein folding involves a stochastic search through the configurational energy landscape towards the native structure. Although most proteins have evolved to fold efficiently into a unique native structure, misfolding (the formation of non-native structures) occurs frequently in vivo causing a wide range of diseases. The prion protein PrP has the unique ability to propagate an infectious disease without transmitting any genetic material, based instead on a misfolded conformation which can reproduce itself. The mechanism of prion misfolding and propagation remains unsettled, from details about the earliest stages of misfolding to the structure of the infectious state. Part of the difficulty in understanding the structural conversion arises from the complexity of the underlying energy landscape. Single-molecule methods provide a powerful tool for probing complex folding pathways as in protein misfolding, because they allow rare and transient events to be observed directly.
  We used custom-built high resolution optical tweezers to study PrP one molecule at a time. By measuring folding trajectories of single PrP molecules held under tension, we found that the native folding pathway involves only two states, without evidence for partially folded intermediates that have been proposed to mediate misfolding. The full energy profile was reconstructed for the native folding of PrP, revealing a double-well potential with an extended partially-unfolded transition state. Interestingly, three different misfolding pathways were detected, all starting from the unfolded state. A mutant PrP with higher aggregation propensity showed increased occupancy of some of the misfolded states, suggesting these states may act as intermediates during aggregation. To investigate the mechanism of PrP misfolding further, we characterized the folding pathways of PrP when two molecules interact to form a dimer. Remarkably, the dimer invariably formed a stable misfolded structure, via multiple partially-folded intermediates. We mapped the energy landscape for PrP dimer misfolding and identified a key intermediate that leads to misfolding by kinetically blocking the formation of the native structure. These results provide mechanistic insight into the formation of non-native structures of PrP and demonstrate a general platform for studying protein misfolding and aggregation at the single-molecule level, with wide applicability for understanding disease and biological function.

• Subjects / Keywords

  • Prion diseases
  • Single molecule
  • Energy landscape
  • Protein misfolding
  • Aggregation
  • Prion protein
  • Riboswitch
  • Energy-landscape reconstruction
  • Biophysics
  • RNA folding
  • Transition path time
  • Transmissible spongiform encephalopathies
  • Kinetics
  • Opitcal tweezers
  • Tandem dimer
  • Force spectroscopy
  • Protein folding

• Graduation date

  Fall 2013

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3K06X87J

• 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.