Crucial biological processes are often dictated by the action of proteins, including cell growth, intercellular communication and apoptosis. The behaviour and function of proteins, and other important bio-macromolecules, is inherently linked to the 3D con-formation of the polypeptide and the range of dynamics within the structure. The rapidly developing tool, solid-state NMR, is uniquely placed to structurally probe large, non-crystalline macromolecules, such as proteins, and provide data at atomic-level resolution. Amyloid diseases, such as Alzheimer's disease and Parkinson's disease, are linked to neuronal damage caused by toxic species which occur through misfolding and aggregation of naturally expressed proteins. Aggregation of the 36-43 residue protein amyloid-beta (A ) is thought to be involved in the pathology of Alzheimer's disease. In this thesis, solid-state NMR is used to obtain 1 H,15 N and 13 C chemical shifts for U-[13 C,15 N] A 1-40 aggregates formed in the presence of copper, a metal found in abnormally high concentrations in amyloidogenic plaques in Alzheimer's-disease-afflicted patients' brains via post-mortem examination. A suite of 2D13 C-13 C DARR and15 N-13 C DCP experiments alongside 2D and 3D X-1 H,15 N-1 H-1 H and13 C-15 N-1 H. Inverse Detection experiments employed at 100 kHz MAS frequency is employed to obtain chemical shift values. TALOS-N provides torsion angle restraints and secondary chemical shift analysis is performed to identify turn and -sheet regions. The results are compared to previously published models, and indicate the sample is similar to a brain-derived fibrillar structure. 2D13 C-13 C DARR experiments are also performed on selectively labelled samples of A 1-42 with cysteine replacements at locations A21 and A30. This cross-linking mechanism between C21 and C30 has been shown to stabilise an oligomeric form, thought to be the most toxic, on the aggregatory pathway to fibrils. The labels allow detailed characterisation of the turn region between residues 22 and 29. The incorporation of the oligomers into lipid membranes was studied by 1D31 P NMR, specifically to investigate the effect of the oligomers on the stability of the membranes and the effect of cholesterol and curcumin, a potentially therapeutic compound currently of high interest, on this de-stabilisation. Work with collaborators has enabled the oligomeric form to be modelled and the results indicate these toxic oligomers form a hexamer barrel structure, with the hydrophobic sidechains contained within the barrel. The secondary structure of the oligomer is shown to contain 3 or 4 -sheets; with an extended -sheet region between K16-V24, 1 or 2 -sheets between N27 and V36, and then a short -sheet between V39 and A42. A method for inter-molecular –sheet interaction in the hexamer is postulated. A method for further aggregation from this oligomeric form to a protofibrillar form has also been suggested, thus enabling a claim to be made for an on-pathway nature for this oligomeric form.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:655614 |
| Date | January 2015 |
| Creators | Kelly, Robert Thomas |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/68832/ |
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