A variety of experimental approaches implicate the ordered aggregation of the Abeta peptide in the cortex as a key step in the progressive loss of neuronal function characteristic of Alzheimer's disease. Current thinking favors a toxic role for rapidly formed aggregates, called protofibrils, of Abeta peptides ending at residue 41 to 43. However, it is also possible that mature amyloid fibrils composed of these peptides, as well as shorter versions ending at positions 39 or 40, may also influence disease, as either toxic or protective entities. Although our knowledge of Abeta amyloid structure is at present only at modest resolution, great strides have been made in the last four years in the analysis of the structure and structural energetics of Abeta aggregates, and new tools for structural analysis of aggregates have been developed. This proposal builds on previous work by focusing on (a) the structure and stabilization of Abeta (1-42) amyloid;(b) the structure and stabilization of Abeta (1-42) protofibrils;and (c) the structure and properties of different conformational types of Abeta amyloid fibrils recently reported. Alanine and proline mutants will be aggregated, and the change in stability of amyloid fibrils and protofibrils formed from these mutants will be determined and analyzed. Single and double cysteine mutant crosslinking analysis will be conducted on 1-42 mutants, to determine the internal structure of the fibrils. These and other methods will also be applied to the analysis of the structure and stability of alternative conformations of the fibrils (altered conformational forms of Abeta fibrils may have influenced their biological activity, as is the case for amyloid forms of yeast prions). Involving collaborations with several molecular modeling groups, this proposal will use experimental data to test current models for fibril and protofibril structure and assembly, and will allow for design of additional experiments to test structural models as they are developed. These studies should contribute to the understanding of Abeta amyloid fibril structure, assembly, and the fundamental basis of amyloid fibril structural stabilization, which will be important in developing structural models for amyloid fibrils of Abeta and other peptides responsible for neurodegenerative diseases. In addition, these studies should continue to push the frontiers of our knowledge of Abeta protofibrils and their role in disease. This data will enhance our knowledge of the molecular basis of AD and in the process contribute to the development of molecular targets for drug design.