Fluorescent non-canonical amino acid as a site-specific conformational probe of prion formation

The pathogenic conversion of the cellular prion protein (PrP ^C ) into the β-sheet-rich isoform PrP ^Sc is the pivotal pathogenic event in prion disease, yet the molecular steps that govern this structural transition remain elusive. In this study, we introduce a new approach to monitor site-specific conformational transitions that occur during infectious prion formation. The method relies on genetically encoded substitution of a fluorescent, environmentally sensitive non-canonical amino acid, L-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA), into recombinant PrP substate molecules, allowing real-time monitoring of structural changes in high-efficiency in vitro PrP ^Sc conversion reactions. As proof of principle, we show that the W99 7-HCAA recPrP substate efficiently propagates two different PrP ^Sc conformers (infectious cofactor PrP ^Sc and non-infectious protein-only PrP ^Sc ). Bioassays in knock-in mice expressing bank vole (BV) PrP confirm that W99 7-HCAA cofactor PrP ^Sc produced by serial propagation is infectious, causing scrapie with an incubation period and neuropathological profile like those induced by wild-type cofactor PrP ^Sc . Marked differences in fluorescence intensity were observed between native, misfolded, and denatured states of W99 7-HCAA PrP, confirming that 7-HCAA reports on local changes in PrP conformation. Together, these findings establish 7-HCAA as a site-specific and sensitive probe of local PrP conformation. Moreover, the results suggest a new and broadly applicable strategy for studying conformational dynamics in amyloid-forming proteins.