Self-Assembly of Recombinant Prion Protein of 106 Residues

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Abstract
The central event in the pathogenesis of prion diseases is a profound conformational change of the prion protein (PrP) from an α-helical (PrPC) to a β-sheet-rich isoform (PrPSc). The elucidation of the mechanism of conformational transition has been complicated by the challenge of collecting high-resolution biophysical data on the relatively insoluble aggregation-prone PrPSc isoform. In an attempt to facilitate the structural analysis of PrPSc, a redacted chimeric mouse-hamster PrP of 106 amino acids (MHM2 PrP106) with two deletions (Δ23−88 and Δ141−176) was expressed and purified from Escherichia coli. PrP106 retains the ability to support PrPSc formation in transgenic mice, implying that it contains all regions of PrP that are necessary for the conformational transition into the pathogenic isoform [Supattapone, S., et al. (1999) Cell 96, 869−878]. Unstructured at low concentrations, recombinant unglycosylated PrP106 (rPrP106) undergoes a concentration-dependent conformational transition to a β-sheet-rich form. Following the conformational transition, rPrP106 possesses properties similar to those of PrPSc106, such as high β-sheet content, defined tertiary structure, resistance to limited digestion by proteinase K, and high thermodynamic stability. In GdnHCl-induced denaturation studies, a single cooperative conformational transition between the unstructured monomer and the assembled β-oligomer was observed. After proteinase K digestion, the oligomers retain an intact core with unusually high β-sheet content (>80%). Using mass spectrometry, we discovered that the region of residues 134−215 of rPrP106 is protected from proteinase K digestion and possesses a solvent-independent propensity to adopt a β-sheet-rich conformation. In contrast to the PrPSc106 purified from the brains of neurologically impaired animals, multimeric β-rPrP106 remains soluble, providing opportunities for detailed structural studies.