Prions are composed largely, if not entirely, of prion protein (PrPsc in the case of scrapie). Although the formation of PrPs from the cellular prion protein (PrPc) is a post-translational process, no candidate chemical modification was identified, suggesting that a conformational change features in PrPsc synthesis. To assess this possibility, we purified both PrPC and PrPsc by using nondenaturing procedures and determined the secondary structure ofeach. Fourier-transform infrared (FTIR) spectroscopy demonstrated that PrPC has a high a-helix content (42%) and no (3sheet (3%), findings that were confirmed by circular dichroism measurements. In contrast, the -sheet content of PrPSc was 43% and the a-helix

Because the insolubility of the scrapie prion protein (PrP Sc ) has frustrated structural studies by x-ray crystallography or NMR spectroscopy, we used electron crystallography to characterize the structure of two infectious variants of the prion protein. Isomorphous two-dimensional crystals of the N-terminally truncated PrP Sc (PrP 27-30) and a miniprion (PrP Sc 106) were identified by negative stain electron microscopy. Image processing allowed the extraction of limited structural information to 7 Å resolution. By comparing projection maps of PrP 27-30 and PrP Sc 106, we visualized the 36-residue internal deletion of the miniprion and localized the N-linked sugars. The dimensions of the monomer and the locations of the deleted segment and sugars were used as constraints in the construction of models for PrP Sc . Only models featuring parallel ␤-helices as the key element could satisfy the constraints. These low-resolution projection maps and models have implications for understanding prion propagation and the pathogenesis of neurodegeneration.electron microscopy ͉ image processing ͉ Nanogold labeling ͉ parallel ␤-helix ͉ amyloid structure C reutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy (BSE), scrapie, and other spongiform encephalopathies are caused by an aberrantly folded isoform (PrP Sc ) of the prion protein (PrP) (1). Replication of prions includes a profound change in the conformation of the cellular isoform of PrP (PrP C ) to form the highly insoluble PrP Sc . The insolubility of PrP Sc has thwarted attempts to investigate its structure by either x-ray crystallography or NMR spectroscopy. Our knowledge about the structure of PrP Sc is therefore rather limited (2).After treatment with proteinase K (PK), PrP Sc loses the N-terminal residues 23 to Ϸ89 (forming PrP 27-30), but retains infectivity. During purification, PrP 27-30 polymerizes into rod-shaped filaments with the tinctorial properties of amyloid (3, 4). X-ray fibril diffraction illustrated the amyloid nature of PrP 27-30; characteristic 4.7 Å reflections indicative of cross-␤ structure were observed (5). Optical spectroscopy revealed that PrP Sc and PrP 27-30 are substantially enriched in ␤-sheet structure (6-9). This finding is in sharp contrast to the predominantly ␣-helical fold of the three-helix-bundle structure of PrP C as determined by NMR spectroscopy and x-ray crystallography on refolded recombinant PrP (10 -18). Owing to the lack of high-resolution structural information for PrP Sc , predictive methods have been used to develop molecular models to codify the existing spectroscopic, immunological, and biochemical data (19).In attempts to simplify the structural analysis of PrP Sc , we systematically deleted parts of the prion protein. One of these constructs containing only 106 residues, PrP106 (⌬23-88, ⌬141-176), supported the propagation of prions (20,21). Transgenic mice expressing only PrP106 develop a histologically accurate neurodegenerative prion disease after inoculation with prions, and the resulting prions can...

With the discovery of the prion protein (PrP), immunodiagnostic procedures were applied to diagnose Creutzfeldt-Jakob disease (CJD). Before development of the conformation-dependent immunoassay (CDI), all immunoassays for the disease-causing PrP isoform (PrP Sc ) used limited proteolysis to digest the precursor cellular PrP (PrP C ). Because the CDI is the only immunoassay that measures both the protease-resistant and protease-sensitive forms of PrP Sc , we used the CDI to diagnose human prion disease. The CDI gave a positive signal for PrP Sc in all 10 -24 brain regions (100%) examined from 28 CJD patients. A subset of 18 brain regions from 8 patients with sporadic CJD (sCJD) was examined by histology, immunohistochemistry (IHC), and the CDI. Three of the 18 regions (17%) were consistently positive by histology and 4 of 18 (22%) by IHC for the 8 sCJD patients. In contrast, the CDI was positive in all 18 regions (100%) for all 8 sCJD patients. In both gray and white matter, Ϸ90% of the total PrP Sc was protease-sensitive and, thus, would have been degraded by procedures using proteases to eliminate PrP C . Our findings argue that the CDI should be used to establish or rule out the diagnosis of prion disease when a small number of samples is available as is the case with brain biopsy. Moreover, IHC should not be used as the standard against which all other immunodiagnostic techniques are compared because an immunoassay, such as the CDI, is substantially more sensitive.

Mice, homozygous for prion protein (PrP) gene ablation (Prn-p°/°), develop normally and remain well >500 days after inoculation with murine scrapie prions. In contrast, wild-type mice developed scrapie <165 days after inoculation and most Prn-p°/+ mice, heterozygous for disrup-