Prion diseases result from the accumulation of a misfolded isoform (PrP Sc ) of the normal host prion protein (PrP C ). PrP Sc propagates by templating its conformation onto resident PrP C to generate new PrP Sc . Although the nature of the PrP Sc -PrP C complex is unresolved, certain segments or specific residues are thought to feature critically in its formation. The polymorphic residue 129 is one such site under considerable study. We combined transmission studies with a novel live cell yeast-based fluorescence resonance energy transfer (FRET) system that models the molecular association of PrP in a PrP Sc -like state, as a way to explore the role of residue 129 in this process. We show that a reduction in efficiency of prion transmission between donor PrP Sc and recipient PrP C that are mismatched at residue 129 correlates with a reduction in FRET between PrP-129M and PrP-129V in our yeast model. We further show that this effect depends on the different secondary structure propensities of Met and Val, rather than the specific amino acids. Finally, introduction of the disease-associated P101L mutation (mouseequivalent) abolished FRET with wild-type mouse PrP, whereas mutant PrP-P101L displayed high FRET with homologous PrP-P101L, as long as residue 129 matched. These studies provide the first evidence for a physical alteration in the molecular association of PrP molecules differing in one or more residues, and they further predict that the different secondary structure propensities of Met and Val define the impaired association observed between PrP Sc and PrP C mismatched at residue 129.The prion diseases are transmissible neurodegenerative disorders that result from the accumulation of a misfolded isoform (PrP Sc ) of the normally folded cellular prion protein (PrP C ). The former is distinguished from the latter by its insolubility in nonionic detergents and relative resistance to proteinase K (PK) 2 digestion (1). Once generated, PrP Sc propagates by templating its conformation onto PrP C , leading to the accumulation of PrP Sc and associated central nervous system pathologic features of neuronal death, gliosis, and vacuolation (2). The PrP ScPrP C interface has not been resolved, although a specific orientation is predicted, based on several lines of evidence suggesting that sequence homology between PrP Sc and PrP C at key sites within the molecule are necessary for effective propagation of PrP Sc , a feature that forms the basis for the well recognized species barrier to prion transmission (3).The central region of PrP has been shown to feature prominently in defining the species barrier (4), and within the first -strand in this central region lies residue 129, a polymorphic site that plays a key role in disease risk and phenotype determination (5, 6). Compared with the general population, homozygosity for either Met or Val is significantly more prevalent in Creutzfeldt-Jakob disease (CJD) (5), whereas the course of disease in 129MV patients is generally more protracted (7,8). Furthermore, all primary ...