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Title page for ETD etd-03112014-135805

Type of Document Dissertation
Author Wan, William Nicholas
URN etd-03112014-135805
Title Structure and Assembly of the Fungal Prion-forming Domain HET-s(218-289)
Degree PhD
Department Chemical and Physical Biology
Advisory Committee
Advisor Name Title
Charles R Sanders Committee Chair
Anne K Kenworthy Committee Member
Benjamin Spiller Committee Member
Martin Egli Committee Member
  • amyloid
  • self-propagation
  • fiber diffraction
  • prion
Date of Defense 2014-03-07
Availability unrestricted
Prions are infectious proteins; aberrantly folded proteins with self-propagating structures that

induce a biological effect. Prions are implicated in a number of diseases, including the transmissi-

ble spongiform encephalopathies (TSEs), a family of diseases that includes scrapie of sheep, bovine

spongiform encephalopathy, chronic wasting disease in deer, and Creutzfeldt-Jakob disease in humans. The prions found in each of these diseases consist of a single protein, PrP, and take the form of amyloids, filamentous protein aggregates with cross-β structure. Despite consisting of a single protein, prions can take on different folds, each producing a distinct phenotype; this is known as the strain phenomenon. In addition to prion diseases, amyloids are also implicated in many other diseases including Alzheimer’s disease, Parkinson’s disease, chronic trauma encephalopathy, and type II diabetes. It is becoming apparent that these diseases have prion-like properties; pathology can be induced through the introduction of exogenous amyloid. Through exploiting the self-propagating and self-assembling properties of cross-β structures, some organisms have evolved functional prions and amyloids. The biological activities of prions and amyloids are determined by their aberrant folds, making structural insights into these folds key to understanding their mechanisms of self-propagation and self-assembly.

In this work, we made use of the functional fungal prion-forming domain HET-s(218–289) in order

to study prion polymorphism and self-propagation. HET-s(218–289) forms an infectious β-solenoid

fold at physiological pH, but forms non-infectious fibrils under acidic conditions. We characterized the structural differences between HET-s(218–289) polymorphs and found that the fibrils formed at acidic pH had stacked β-sheet architectures, which tend to represent generic low-energy states. Stacked β-sheets were unable to propagate their structure at physiological pH, but did modify fibrillization kinetics, demonstrating heterogeneous seeding, a process where one structure nucleates the fibrillization of another. Using site-directed mutagenesis, we characterized the biophysical properties of various structural features of the HET-s(218–289) β-solenoid fold, including fibril architecture, fibrillization kinetics, and chemical stability. Our results elucidated some of the structural features that provide HET-s(218–289) with robust self-assembling properties, as well as the relative importance of each type of structural feature.

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