(631h) Characterization of Prion Protein Self-Interactions By Yeast Surface Display and Next-Generation Sequencing

Prion diseases are propagated by the conversion of the native form of the prion protein (PrP^C) to an alternatively folded, disease causing form (PrP^Sc), catalyzed by the interaction of PrP^Sc with PrP^C.  In order to characterize prion protein self-interactions we expressed mouse prion protein on the yeast surface and investigated its interactions with anti-prion antibodies, recombinant prion protein, and brain-derived prions. Epitope mapping of anti-prion antibodies provided residue level resolution of conformational epitopes, suggesting that the prion protein exists in a structure similar to the native conformation when expressed on the yeast surface. Using the yeast surface display platform, we observed that the affinity of prion self-interaction was highly dependent on protein conformation; showing low affinity for recombinant prion protein (recPrP) in an alpha-helical structure and higher affinities toward beta sheet rich structures including soluble beta-sheet rich recPrP, synthetic recPrP amyloids and brain derived PrP^Sc. In order to identify key residues responsible for molecular recognition, we employed a mutational scanning and deep sequencing approach. A 8.9 x 10⁵ member random mutant prion protein library tethered to the yeast surface was constructed by error prone PCR to introduce mutations at each amino acid position. The library was incubated with soluble, fluorescently labeled antibodies and prion mutants with reduced affinity for various prion protein conformations were isolated by florescence-activated cell sorting (FACS). Sequencing of the selected population using single molecule real time (SMRT) genetic sequencing identified residues critical for prion protein self-interaction. Identification of residues responsible for prion protein self-interaction provides molecular insight into the replicative interface of prions and disease pathogenesis.