Self-assembly of recombinant prion protein of 106 residues

The central event in the pathogenesis of prion diseases is a profound confo rmational change of the prion protein (PrP) from an alpha-helical (PrPC) to a beta-sheet-rich isoform (PrPSc). The elucidation of the mechanism of con formational transition has been complicated by the challenge of collecting high-resolution biophysical data on the relatively insoluble aggregation-pr one PrPSc isoform. In tin attempt to facilitate the structural analysis of PrPSc, a redacted chimeric mouse-hamster PrP of 106 amino acids (MHM2 PrP10 6) with two deletions (Delta 23-88 and Delta 141-176) was expressed and pur ified from Escherichia coli. PrP106 retains the ability to support PrPSc fo rmation in transgenic mice, implying that it: contains all regions of PrP t hat are necessary for the conformational transition into the pathogenic iso form [Supattapone, S., et al. (1999) Cell 96, 869-878]. Unstructured at low concentrations, recombinant unglycosylated PrP106 (rPrP106) undergoes a co ncentration-dependent conformational transition to a beta-sheet-rich form. Following the conformational transition, rPrP106 possesses properties simil ar to those of PrP(Sc)106, such as high beta-sheet content, defined tertiar y structure, resistance to limited digestion by proteinase K, and high ther modynamic stability. In GdnHCl-induced denaturation studies, a single coope rative conformational transition between the unstructured monomer and the a ssembled beta-oligomer was observed. After proteinase K digestion, the olig omers retain an intact core with unusually high beta-sheet content (>80%). Using mass spectrometry, we discovered that the region of residues 134-215 of rPrP106 is protected from proteinase K digestion anti possesses a solven t-independent propensity to adopt a beta-sheet-rich conformation. Tn contra st to the PrP(Sc)106 purified from the brains of neurologically impaired an imals, multimeric beta-rPrP106 remains soluble, providing opportunities for detailed structural studies.