1) We continued to probe the structures of the infectious form of prion protein, PrPSc. The prevalent structural models of PrPSc retain most of the native alpha-helices of the normal, non-infectious prion protein, PrPC, but evidence is accumulating that these helices are absent in PrPSc amyloid. We obtained solid-state NMR evidence that prion-seeded recombinant PrP amyloids formed in vitro in the absence of denaturants have parallel in-register intermolecular beta-sheet architectures in the domains originally occupied by helices 2 and 3. These results, in the context of a primarily beta-sheet structure, led us to build detailed models of PrP amyloid based on parallel in-register architectures, fibrillar shapes and dimensions, and other available experimentally derived conformational constraints. Molecular dynamics simulations of PrP 90-231 octameric segments suggested that such linear fibrils, which are consistent with many features of PrPSc fibrils, can have stable parallel in-register beta-sheet cores. These simulations revealed that the C-terminal residues 124-227 more readily adopt stable tightly packed structures than the N-terminal residues 90-123 in the absence of cofactors. Variations in the placement of turns and loops that link the beta-sheets could give rise to distinct prion strains capable of faithful template-driven propagation. Moreover, our modeling suggested that single PrP monomers can comprise the entire cross-section of fibrils that have previously been assumed to be pairs of laterally associated protofilaments. Together these insights provided a new basis for deciphering mammalian prion structures. 2)We have continued to probe the role of normal prion protein in the experimental autoimmune encephalomyelitis, and therapeutic treatments thereof with amyloid fibrils. In doing so, we have found that the genetic background of the mice plays a key role in both PrP involvement and responses to amyloid treatments.
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