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Title: A chemical biology approach to investigate amyloid formation
Author: Valette, Nathalie Marie
ISNI:       0000 0004 2718 6300
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2010
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Many peptides and proteins have now been linked with 'amyloidosis' disorders despite having apparently different structural and functional properties in their native states. Although progress has been made over recent years, it is still unclear how such different proteins can assemble into the common type of cross-B architecture that characterises such aggregates. Detailed knowledge of the mechanism leading to protein misfolding and aggregation, and of the structures of amyloid fibrils is highly important in order to develop new drugs capable of treating these life-threatening diseases. In addition such studies will illuminate the fundamental principles underpinning the behaviour of the peptide chain. Using a chemical approach, modifications that are generally difficult to access with standard biological techniques can be easily introduced into the polypeptide chain. These chemical modifications which can include completely unnatural functionalities can allow site-specific interactions to be studied during fibrillar assembly, and provide a deeper understanding of the underlying causes of aggregation at both peptide and protein scale. In this work we show how the aggregation of a peptide corresponding to strand E of P2-microglobulin can be modulated by phosphorylation. Peptides equivalent to strand E were synthesised with inclusion of phosphoamino acids. By monitoring the aggregation propensity of an array of differentially phosphorylated sequences at a range of pH values, the effect of the phosphate group was determined. Not only was the aggregation pH-dependent, but also the position of the phosphate group in the peptide sequence was important for the propensity of the peptide to aggregate. These results were combined with molecular dynamics simulations to produce models of putative fibrillar architectures. Because small peptide model systems cannot always mimic the aggregation behaviour of the equivalent full-length protein, a synthetic strategy to chemically prepare full-length P2M was developed with the scope to introduce non-natural amino acids and to probe the effect of those on the aggregation propensity of P2M. A four fragment strategy previously developed in the group with a single point mutation at Ser52 and a phosphotyrosine residue at position 67 was optimised and enhanced, and the conditions to achieve the final folding and oxidation steps were determined. A synthetic route to prepare a true wild-type P2M requiring a 55-amino acid long peptide fragment was also developed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available