Detailed biophysical characterization of the acid-induced PrP(c) to PrP(β) conversion process

Prions are believed to spontaneously convert from a native, monomeric highly helical form (called PrP(c)) to a largely β-sheet-rich, multimeric and insoluble aggregate (called PrP(sc)). Because of its large size and insolubility, biophysical characterization of PrP(sc) has been difficult, and there are several contradictory or incomplete models of the PrP(sc) structure. A β-sheet-rich, soluble intermediate, called PrP(β), exhibits many of the same features as PrP(sc) and can be generated using a combination of low pH and/or mild denaturing conditions. Studies of the PrP(c) to PrP(β) conversion process and of PrP(β) folding intermediates may provide insights into the structure of PrP(sc). Using a truncated, recombinant version of Syrian hamster PrP(β) (shPrP(90-232)), we used NMR spectroscopy, in combination with other biophysical techniques (circular dichroism, dynamic light scattering, electron microscopy, fluorescence spectroscopy, mass spectrometry, and proteinase K digestion), to characterize the pH-driven PrP(c) to PrP(β) conversion process in detail. Our results show that below pH 2.8 the protein oligomerizes and conversion to the β-rich structure is initiated. At pH 1.7 and above, the oligomeric protein can recover its native monomeric state through dialysis to pH 5.2. However, when conversion is completed at pH 1.0, the large oligomer "locks down" irreversibly into a stable, β-rich form. At pH values above 3.0, the protein is amenable to NMR investigation. Chemical shift perturbations, NOE, amide line width, and T(2) measurements implicate the putative "amylome motif" region, "NNQNNF" as the region most involved in the initial helix-to-β conversion phase. We also found that acid-induced PrP(β) oligomers could be converted to fibrils without the use of chaotropic denaturants. The latter finding represents one of the first examples wherein physiologically accessible conditions (i.e., only low pH) were used to achieve PrP conversion and fibril formation.