Triangulating from the cairns…
The conversion of the normal form of prion protein (PrPC) to a disease-specific form (PrPSc) is a central event in scrapie and other transmissible spongiform encephalopathies. PrPSc is distinguished from PrPC by its insolubility and its resistance to proteolysis. PrPSc is also capable of converting 35S-PrPC, in vitro, into a form which is indistinguishable from PrPSc with respect to its protease-sensitivity. Both the "converting activity" and the protease-resistance of isolated hamster PrPSc can be at least partially eliminated by denaturation and recovered by renaturation, provided that the concentration of denaturant does not exceed a threshhold. This study was undertaken in order to localize 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 fragment marked by an epitope within residues 143-156 remained protease-resistant under conditions which at least partially unfolded epitopes within residues 90-115 and 217-232. However, dilution of denaturant restored protease-resistance to these epitopes. This reversible unfolding was observed with both purified PrPSc and PrPSc in crude brain homogenates. Size fractionation of partially GdnHCl-solubilized PrPSc revealed that only the insoluble aggregates retained the ability to refold, consistent with the hypothesis that native PrPSc is an ordered aggregate. When the threshold denaturant concentration was exceeded, both protease-resistance of the 16-kDa C-terminal domain and converting activity were irreversibly destroyed. These results suggest that the in vitro converting activity requires ordered, protease-resistant PrPSc aggregates and that a critical aspect of the PrPSc structure is the folding of a particularly stable approximately 16-kDa C-terminal domain.