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Title: Studies on the mechanism of amyloid formation by cystatin B
Author: Paramore, Robert
ISNI:       0000 0004 2742 2721
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2010
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Many diseases, including Alzheimer's and Creutzfeldt-Jakob disease, are believed to be the result of protein misfolding and aggregation into insoluble, highly ordered Β-sheet structures known as amyloid fibrils. The mechanism of formation of these fibrils is unknown. The work presented in this thesis uses human cystatin B as a model system for the elucidation of the mechanism of fibril formation. Cystatin B, a cysteine protease inhibitor, readily forms amyloid fibrils in vitro. Amyloid fibrils formed from the structurally homologous cystatin C, are associated with a form of cerebral haemorrhage as well as incorporation in amyloid derived from Alzheimer's disease sufferers. The work in this thesis sheds light on the mechanism by which proteins spontaneously change conformation and self associate to form amyloid fibrils. Investigation of the state induced by conditions of fibrillisation and the effects of single point mutations highlight particular regions within the natively folded protein that are involved in the self association process. Studies on Cu2+ and Zn2+ binding to cystatin B highlight the same small regions of importance and show that the effect induced by divalent metals is very metal specific. It is also shown that fibrillisation of cystatin B is preceded by aggregation into a large insoluble structure. This may act to increase local concentration by protein adsorption and involve unknown surface interactions that induce or catalyse the initiation of fibrillisation. The interactions and conformational changes required for fibrillisation are likely to be local rather than global events which require the weakening of the co-operative properties normally associated with protein folding. Alteration of these interactions will promote alternative pathways of assembly resulting in different "strains" of amyloid. Ultimately the high resolution structure of cystatin B fibrils will answer many questions and the initial solid state NMR experiments to achieve this are presented in this thesis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available