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

de Alcantara Ferreira, J.; Walsh, D. J.; Turnbaugh, E.; Mills, J. H.; Supattapone, S.

doi:10.64898/2026.04.21.719968

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bioRxiv · 10.64898/2026.04.21.719968

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

de Alcantara Ferreira, J.·Walsh, D. J.·Turnbaugh, E.·Mills, J. H.·Supattapone, S.

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Abstract

The pathogenic conversion of the cellular prion protein (PrPC) into the {beta}-sheet-rich isoform PrPSc 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 PrPSc conversion reactions. As proof of principle, we show that the W99 7-HCAA recPrP substate efficiently propagates two different PrPSc conformers (infectious cofactor PrPSc and non-infectious protein-only PrPSc). Bioassays in knock-in mice expressing bank vole (BV) PrP confirm that W99 7-HCAA cofactor PrPSc produced by serial propagation is infectious, causing scrapie with an incubation period and neuropathological profile like those induced by wild-type cofactor PrPSc. 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.

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de Alcantara Ferreira, J., Walsh, D. J., Turnbaugh, E., Mills, J. H., Supattapone, S.. 2026-04-24. Fluorescent non-canonical amino acid as a site-specific conformational probe of prion formation. https://doi.org/10.64898/2026.04.21.719968

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