PROPERTIES OF THE SCRAPIE PRION PROTEIN - QUANTITATIVE-ANALYSIS OF PROTEASE RESISTANCE

The disease-specific isoform of the prion protein (PrPSc) is an essential part of the infectious particle which causes spongiform degeneration in various mammalian species. PrPSc differs from PrP of normal animals (PrPC) by its relative protease resistance. The physical nature of this difference is still unknown. We analyzed the protease resistance of PrPSc quantitatively using an enzyme-linked immuno-filtration assay. PrPSc was rendered completely protease-sensitive at alkaline pH or in >1.5 M guanidinium thiocyanate (GdnSCN). Denaturation in 4 M GdnSCN completely abolished the protease resistance of PrPSc within 15 min, while denaturation in 7.2 M urea showed a slower time course. In the presence of ethanol, PrPSc was protected from denaturation by GdnSCN or alkaline pH. Denaturation curves were used to calculate the free energy (Delta G(D)) as a function of different denaturant concentrations. Linear regression of Delta G(D) values was used to extrapolate the free energy in the absence of denaturants (Delta G(H2O)>, yielding similar values (Delta G(H2O,GdnSCN)=-2.3 kcal/mol;Delta G(H2O,urea)=-3.1 kcal/mol). The linear relationship between Delta G(D) and the denaturant concentration is suggestive of a two-state model involving the conformational change of a single protein domain. This is also reflected in the small number of side chains (11.6) additionally exposed to the solvent upon conversion of PrPSc to its protease-sensitive isoform. Our results suggest that only minor rearrangements of the structure of PrP are needed to abolish the protease resistance of PrPSc.