PARTIAL UNFOLDING AND REFOLDING OF SCRAPIE-ASSOCIATED PRION PROTEIN -EVIDENCE FOR A CRITICAL 16-KDA C-TERMINAL DOMAIN

The conversion of the normal form of prion protein (PrPC) to a disease -specific form (PrPSc) is a central event in scrapie and other transmi ssible spongiform encephalopathies, PrPSc is distinguished from PrPC b y its insolubility and its resistance to proteolysis. PrPSc is also ca pable of converting S-35-PrPC, ii? vitro, into a form which is indisti nguishable from PrPSc with respect to its protease-sensitivity. Both t he ''converting activity'' and the protease-resistance of isolated ham ster PrPSc can be at least partially eliminated by denaturation and re covered by renaturation, provided that the concentration of denaturant does not exceed a threshhold. This study was undertaken in order to l ocalize the regions of native PrPSc structure that must remain intact to allow refolding. Proteinase K was used to digest exposed, denatured PrPSc sequences, and the residual fragments were characterized using anti-PrP antibodies directed toward four PrP epitopes. A 16-kDa fragme nt marked by an epitope within residues 143-156 remained protease-resi stant under conditions which at least partially unfolded epitopes with in residues 90-115 and 217-232, However, dilution of denaturant restor ed protease-resistance to these epitopes. This reversible unfolding wa s observed with both purified PrPSc and PrPSc in crude brain homogenat es. Size fractionation of partially GdnHCl-solubilized PrPSc revealed that only the insoluble aggregates retained the ability to refold, con sistent with the hypothesis that native PrPSc is an ordered aggregate, When the threshold denaturant concentration was exceeded, both protea se-resistance of the 16-kDa C-terminal domain and converting activity were irreversibly destroyed. These results suggest that the in vitro c onverting activity requires ordered, protease-resistant PrPSc aggregat es and that a critical aspect of the PrPSc structure is the folding of a particularly stable similar to 16-kDa C-terminal domain.