Detailed biophysical characterization of the acid-induced PrPc to PrPβ conversion process

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DOIResolve DOI: http://doi.org/10.1021/bi101435c
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TypeArticle
Journal titleBiochemistry
ISSN0006-2960
Volume50
Issue7
Pages11621173; # of pages: 12
AbstractPrions 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. © 2010 American Chemical Society.
Publication date
LanguageEnglish
AffiliationNational Research Council Canada (NRC-CNRC); National Institute for Nanotechnology
Peer reviewedYes
NPARC number21271942
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Record identifier070e02f2-0dbe-43f4-92e3-5d04b4e8cc42
Record created2014-05-13
Record modified2016-05-09
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