Determination of the high resolution structure of the polypeptide chain in amyloid fibrils

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Amyloid fibrils are highly organised filamentous structures formed by the self-assembly of polypeptides, and in vivo they are implicated in a number of diseases including Alzheimer¿s Disease and type II diabetes. Despite the fact that many different chemically unrelated proteins form amyloid fibrils, the final fibrillar complexes appear to share similar fundamental structures. The primary objective of the proposed work is to determine the intermolecular organisation of polypeptide in a model amyloid fibril system at high resolution using solid-state NMR techniques. Our model system is an 11-amino acid peptide that readily self-assembles, and we have already had great success in determining the high resolution structure of the individual peptides in their fibrillar form. All that remains is to determine the three-dimensional organisation of the peptides in a fibrillar array. The experimental data will be combined with molecular dynamics simulations to confirm and predict intermolecular constraints, and using this combination of experimental and theoretical approaches we will build up the first high-resolution structure of a system that to date has remained intractable. The second objective of the proposal is to extend the knowledge and methodological experience gained from this model polypeptide system to a significantly larger protein of 84 amino acids, the SH3 domain of the p85a subunit of phosphatidyl-inositol-3 kinase. Such a protein is larger than any system measured by solid-state NMR techniques to date and thus represents a significant challenge. In the first instance, we propose to determine the complete sequence assignments for this protein, an essential first step towards the determination of a structure, and a goal achievable within the lifetime of the proposed project. All of these approaches will provide us with insight into a type of quaternary structure that has singular relevance to a wide range of degenerative diseases.