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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

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.





Author: Yu, Hao

Source: https://era.library.ualberta.ca/


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University of Alberta Single-molecule studies of prion protein folding and misfolding by Hao Yu A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Physics ©Hao Yu Fall 2013 Edmonton, Alberta 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 authors prior written permission. Dedication In memory of my maternal grandfather, Zhipan Qi 1926-2013 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, bec...





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