Prion diseases are a group of transmissible neurodegenerative disorders that include Creutzfeldt-Jakob disease and Gerstmann-Straussler-Scheinker (GSS) disease in humans, scrapie in sheep, bovine spongiform encephalopathy (“mad cow disease”) in cattle and chronic wasting disease in cervids. The most intriguing aspect of these disorders is the nature of the infectious prion pathogen that is believed to be a misfolded protein aggregate with characteristics of an amyloid. However, the structure of these infectious protein particles remains largely unknown. The overall objective of this project is to gain high-resolution structural insight into the mechanism of prion propagation as well as the phenomena of prion strains and transmissibility barriers. To this end, we use a model of amyloid fibrils generated from the C-terminally truncated prion protein PrP23-144, a variant associated with the Y145Stop phenotype of a GSS-like disease. A unique advantage of this model is that it is amenable to detailed structural characterization at atomic level by solid-state nuclear magnetic resonance (NMR) spectroscopy and other biophysical techniques. Three interrelated specific aims are proposed. The first aim is to determine the high-resolution structures for several different strains of mouse and Syrian hamster PrP23-144 amyloid fibrils using solid-state NMR. In combination with the high-resolution structure of human PrP23-144 amyloid already determined by us, these data will be used to gain insight into the structural basis of PrP amyloid seeding specificities, an in vitro surrogate of transmissibility barriers. In the second aim, we will use the PrP23-144 amyloid model to elucidate the poorly understood phenomenon of prion strain switching as well as the mechanism of strain selection. Finally, the third aim seeks to determine high-resolution structures of PrP amyloids associated with distinct phenotypes of GSS disease. The latter insight is of fundamental importance, as no information is at present available regarding the structures of GSS-associated PrP amyloids or the relationship between specific structural features and disease phenotype.