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Permanent link (DOI): https://doi.org/10.7939/R3K06X87J

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Single-molecule studies of prion protein folding and misfolding Open Access

Descriptions

Other title
Subject/Keyword
single molecule
protein misfolding
biophysics
RNA folding
force spectroscopy
prion disease
energy-landscape reconstruction
aggregation
transmissible spongiform encephalopathies
riboswitch
energy landscape
tandem dimer
protein folding
opitcal tweezers
kinetics
transition path time
prion protein
Type of item
Thesis
Degree grantor
University of Alberta
Author or creator
Yu, Hao
Supervisor and department
Woodside, Michael (Physics)
Examining committee member and department
Freeman, Mark (Physics)
Hegmann, Frank (Physics)
Wishart, David (Biological Sciences and Computing Science)
Tuszynski, Jack (Physics and Oncology)
Forde, Nancy (Physics)
Department
Department of Physics
Specialization

Date accepted
2013-08-08T13:45:41Z
Graduation date
2013-11
Degree
Doctor of Philosophy
Degree level
Doctoral
Abstract
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.
Language
English
DOI
doi:10.7939/R3K06X87J
Rights
Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.
Citation for previous publication
Neupane, K., Yu, H., Foster, D. A. N., Wang, F. and Woodside, M. T. Single-molecule force spectroscopy of the add adenine riboswitch relates folding to regulatory mechanism. Nucl Acids Res 39, 7677-7687 (2011).Yu, H., Liu, X., Neupane, K., Gupta, A. N., Brigley, A. M., Solanki, A., Sosova, I. and Woodside, M. T. Direct observation of multiple misfolding pathways in a single prion protein molecule. Proc Natl Acad Sci USA 109, 5283-5288 (2012).Yu, H., Gupta, A. N., Liu, X., Neupane, K., Brigley, A. M., Sosova, I. and Woodside, M. T. Energy landscape analysis of the native folding pathway of the prion protein yields the diffusion constant, transition path time, and rates. Proc Natl Acad Sci USA 109, 14452-14457 (2012).Yu, H., Dee, D. R. and Woodside, M. T. Single-molecule approaches to prion protein misfolding. Prion 7, 140-146 (2013).

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