NR ABAZ
AU Baskakov,I.V.; Aagaard,C.; Mehlhorn,I.; Wille,H.; Groth,D.; Baldwin,M.A.; Prusiner,S.B.; Cohen,F.E.
TI Self-assembly of recombinant prion protein of 106 residues
QU Biochemistry 2000 Mar 14; 39(10): 2792-804
PT journal article
AB The central event in the pathogenesis of prion diseases is a profound conformational change of the prion protein (PrP) from an alpha-helical (PrPc) to a beta-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 (Delta23-88 and Delta141-176) 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 beta-sheet-rich form. Following the conformational transition, rPrP106 possesses properties similar to those of PrPsc106, such as high beta-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 beta-oligomer was observed. After proteinase K digestion, the oligomers 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 and possesses a solvent-independent propensity to adopt a beta-sheet-rich conformation. In contrast to the PrPsc106 purified from the brains of neurologically impaired animals, multimeric beta-rPrP106 remains soluble, providing opportunities for detailed structural studies.
MH Chromatography, Gel; Circular Dichroism; Light; PrPc Proteins/chemistry/*genetics/*metabolism/ultrastructure; PrPsc Proteins/chemistry/*genetics/*metabolism/ultrastructure; Protein Conformation; Protein Structure, Secondary/genetics; Recombinant Proteins/chemistry/*metabolism/ultrastructure; Scattering, Radiation; Sequence Deletion; Spectrometry, Fluorescence; Support, Non-U.S. Gov't; Support, U.S. Gov't, P.H.S.; Virus Assembly/genetics
AD Institute for Neurodegenerative Diseases, Department of Neurology, University of California, San Francisco 94143, USA
SP englisch
PO USA