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 immunofiltration 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 GD) as a function of different denaturant concentrations. Linear regression of delta GD values was used to extrapolate the free energy in the absence of denaturants (delta GH2O), yielding similar values (delta GH2O,GdnSCN = -2.3 kcal/mol; delta GH2O,urea = -3.1 kcal/mol). The linear relationship between delta GD 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.
Oesch
B
B
Brain Research Institute, University of Zürich, Switzerland.
Jensen
M
M
Nilsson
P
P
Fogh
J
J
D000818:Animals; D006224:Cricetinae; D010450:Endopeptidases / Q000378:metabolism*; D000431:Ethanol / Q000494:pharmacology; D007124:Immunoenzyme Techniques; D008956:Models, Chemical; D018620:PrPSc Proteins; D011328:Prions / Q000737:chemistry / Q000378:metabolism*; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D012608:Scrapie / Q000378:metabolism; D013816:Thermodynamics
D018620:PrPSc Proteins; D011328:Prions; D000431:Ethanol; D010450:Endopeptidases