Award details

The moleculecular structure of cystatin amyloid fibrils

Reference	BB/C504035/1
Principal Investigator / Supervisor	Dr Rosemary Ann Staniforth
Co-Investigators / Co-Supervisors	Professor Jon Waltho
Institution	University of Sheffield
Department	Molecular Biology and Biotechnology
Funding type	Research
Value (£)	195,056
Status	Completed
Type	Research Grant
Start date	01/06/2005
End date	31/05/2008
Duration	36 months

Abstract

The formation of protein deposits observed in amyloid diseases is connected with cellular deterioration on a scale inevitably leading to death. Attempts so far to model this reaction in vitro have been seriously hampered due to the difficulty in defining the molecular structure of the species involved. Current models suffer from a degree of uncertainty that is discouraging to the design of any therapeutic strategy. Part of the problem is that steps in amyloid formation involve partially denatured species, and part is that the endpoint consists of insoluble, fibrous deposits. Our proposal aims to define the molecular structure of cystatin amyloid fibrils to a resolution so far not achieved for fibres from non-peptide precursors. A further benefit of these studies will be an increased understanding of associated aggregation processes. The first part of our programme will examine the extent to which native contacts are preserved within the amyloid fibre and determine whether these form intra-molecularly or are used in the stabilisation of the inter-molecular interfaces that build the multimer. In the latter case, such interactions are consistent with a three-dimensional domain swapping model for fibrillisation. The methodology we have developed and tested for this work includes site-directed insertion of non-native disulphide bonds into the protein scaffold, fibrillisation in the presence of reducing agent (i.e. in the non cross-linked form) followed by re-oxidation in the fibres, and eventually examination of the position and extent to which cross-links have been formed. The second part of the programme will identify new interfaces in the elongated beta-structure which forms the core of amyloid fibrils. We will use a previous solution NMR method which employs a hydrogen exchange labelling strategy to map hydrogen-bonds within the amyloid fibre. Together with the outcome of our disulphide probing experiment, we expect to build a high resolution picture of cystatin fibrils and provide new insight into the processes involved in amyloidogenesis.

Summary

unavailable

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	Ageing, Structural Biology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

I accept the terms and conditions of use (opens in new window)

export PDF file

back to list new search