Amyloid fibrils are found associated with a growing number of human diseases, including several neurodegenerative diseases, the most prevalent of which is Alzheimer's Disease (AD). In each of these diseases, the protein that is the main component of the amyloid fibril is different. Thus, amyloid is a characteristic type of aggregate structure, rather than a particular protein molecule. Indeed, there is some speculation that this amyloid type of aggregate structure may be a protein folding motif that can be accessed by many protein sequences under the right circumstances. Because of its relevance to both human disease and fundamental understanding of protein folding, it is particularly important to know in more detail the folded structure of the amyloid fibril. Amyloid fibrils do not lend themselves to standard techniques for determining protein structure, with the result that we know very little about this structure at the molecular level beyond the fact that these aggregates are rich in beta sheet secondary structure. We have chosen to work on the amyloid associated with AD, composed of the peptide A-beta. In the research proposed here we will use the technique of hydrogen-deuterium exchange (HX) to map the secondary structure of A-beta in the fibril. Hydrogen bonds, such as are found between polypeptide backbone amide hydrogens in beta sheet, protect amide hydrogens from exchange while most other amide hydrogens freely exchange.
The specific aims of this application are to (1) Develop methods for collecting HX data on A-beta incorporated into amyloid fibrils, using both mass spectrometry (MS) and nuclear magnetic resonance (NMR) to quantify levels of exchange, and use these methods to map the protected and exposed amide hydrogens of A-beta in the fibril; (2) carry out similar exchange studies on other A-beta aggregates, like protofibrils, that are implicated in both amyloid assembly and in Abeta aggregate toxicity; (3) use computational methods in concert with the HX data to build, test, and refine models of protofilament and fibril structure; and (4) conduct exchange experiments on other aggregates of A-beta, including fibrils made in vitro from A-beta fragments as well as neuritic plaque cores isolated from human AD brain material. These studies will give us a closer look at the structure of the amyloid fibril, which will allow us to better understand how fibrils grow and how they disrupt human cells and tissue. An improved knowledge of structure will also improved our ability to identify and design therapeutic molecules for inhibiting the growth and toxicity of A-beta amyloid and other aggregates.
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