Robinson, Philip John (2009) The folding, misfolding and aggregation of prions. PhD thesis, University of Warwick.

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Abstract

Prion diseases are a group of fatal neurodegenerative disorders that include Creutzfeldt-Jakob Disease (CJD), Bovine Spongiform Encephalopathy (BSE) and scrapie, which are all associated with the misfolding of the cellular form of the prion protein, PrPC, into the disease associated isoform, PrPSc. This thesis investigates two properties of PrP that may influence the misfolding process; (i) the normal folding mechanism of PrP and (ii) the interactions of PrP with lipid membranes. Firstly, equilibrium folding experiments investigate whether the folding pathway of PrP is influenced by a disease modulating mutation, Q167R, which confers disease resistance. The unfolding of PrPWt is compared to PrPQ167R by monitoring fluorescence and circular dichroism of folding sensitive tryptophan mutants. The results show that the mutation significantly destabilises the protein, which can be rationalised from high resolution structures of PrP. Furthermore, comparison of the folding of mouse and hamster PrP highlights dramatic differences between their folding pathways, which may contribute to the species barrier that is observed in prion disease transmission. The second part of the thesis studies the influence of membrane environments on prion conversion. Firstly, the interaction between PrP and lipid membranes composed of POPC (a zwitterionic phospholipid) and POPS (an anionic phospholipid), are investigated through fluorescence, circular dichroism and centrifugation binding assays. The results show that PrP interacts peripherally with POPC membranes, without significant changes in protein structure. In contrast, high affinity binding to POPS membranes, results in membrane penetration and an increase in β-sheet structure. Furthermore, cryo-electron microscopy reveals that the PrPmembrane interaction disrupts the native vesicle structure and results in the formation of membrane junctions. Finally the morphology and mechanism of growth of prion aggregates on supported lipid bilayers are studied through atomic force microscopy, which shows how the phospholipid content of membranes directs prions down alternative aggregation pathways.

Item Type:	Thesis or Dissertation (PhD)
Subjects:	Q Science > QP Physiology
Library of Congress Subject Headings (LCSH):	Prion diseases -- Research, Protein folding -- Research, Proteins -- Conformation -- Research, Membranes (Biology)
Date:	September 2009
Institution:	University of Warwick
Theses Department:	Department of Biological Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Pinheiro, Teresa
Sponsors:	Biotechnology and Biological Sciences Research Council (Great Britain) (BBSRC)
Extent:	181 leaves : ill., charts
Language:	eng
URI:	http://wrap.warwick.ac.uk/id/eprint/2792

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