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Prion diseases are fatal neurodegenerative diseases caused by the conformational transition of the cellular prion protein (PrP C ) to the abnormal pathological prion protein (PrP Sc ). In this work, the effects of ellagic acid (EA) and pentagalloylglucose (PGG) on prion protein (PrP) fibrillization were investigated. Fluorescence quenching experiments indicated that both EA and PGG could specifically interact with native human PrP with binding affinities of 1.92 × 10 5 and 2.36 × 10 5  L·mol -1 , respectively. Thioflavin-T (ThT) fluorescence assays showed that the binding of EA or PPG could effectively inhibit the nucleation and elongation of PrP fibrilization and reduce the amount of PrP fibrils generated. EA and PGG could also lead to a significant disaggregation of PrP fibrils. Circular dichroism (CD) measurements suggested that EA- or PPG-bound PrP could preserve a higher content of α-helical structures than β-sheet-rich PrP fibrils. The PrP aggregates formed in the presence of EA or PGG showed lower resistance to proteinase K (PK) digestion. Overall, the present work reported the inhibitory effect of EA and PGG on PrP fibrillization. These two natural polyphenols could be potential prodrug molecules for the prevention and treatment of prion diseases., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The pathogenesis of fatal neurodegenerative prion diseases is closely associated with the conversion of α-helix-rich cellular prion protein into β-sheet-rich scrapie form. Pathogenic point mutations of prion proteins usually promote the conformational conversion and trigger inherited prion diseases. The G131V mutation of human prion protein (HuPrP) was identified to be involved in Gerstmann-Sträussler-Scheinker syndrome. Few studies have been carried out to address the pathogenesis of the G131V mutant. Here, we addressed the effects of the G131V mutation on oligomerization and fibrillization of the full-length HuPrP(23-231) and truncated HuPrP(91-231) proteins. The G131V mutation promotes the oligomerization but alleviates the fibrillization of HuPrP, implying that the oligomerization might play a crucial role in the pathogenic mechanisms of the G131V mutant. Moreover, the flexible N-terminal fragment in either the wild-type or the G131V mutant HuPrP increases the oligomerization tendencies but decreases the fibrillization tendencies. Furthermore, this mutation significantly alters the tertiary structure of human PrPC and might distinctly change the conformational conversion tendency. Interestingly, both guanidine hydrochloride denaturation and thermal denaturation experiments showed that the G131V mutation does not significantly change the thermodynamic stabilities of the HuPrP proteins. This work may be of benefit to a mechanistic understanding of the conformational conversion of prion proteins and also provide clues for the prevention and treatment of prion diseases., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In prion disease, the abnormal conformer of the cellular prion protein, PrP(Sc), deposits in fibrillar protein aggregates in brain and other organs. Limited exposure of PrP(Sc) to proteolytic digestion in vitro generates a core fragment of 19-21 kDa, named PrP27-30, which is also found in vivo. Recent evidence indicates that abnormal truncated fragments other than PrP27-30 may form in prion disease either in vivo or in vitro. We characterized a novel protease-resistant PrP fragment migrating 2-3 kDa faster than PrP27-30 in Creutzfeldt-Jakob disease (CJD) brains. The fragment has a size of about 18.5 kDa when associated with PrP27-30 type 1 (21 kDa) and of 17 kDa when associated with type 2 (19 kDa). Molecular mass and epitope mapping showed that the two fragments share the primary N-terminal sequence with PrP27-30 types 1 and 2, respectively, but lack a few amino acids at the very end of C terminus together with the glycosylphosphatidylinositol anchor. The amounts of the 18.5- or 17-kDa fragments and the previously described 13-kDa PrP(Sc) C-terminal fragment relatively to the PrP27-30 signal significantly differed among CJD subtypes. Furthermore, protease digestion of PrP(Sc) or PrP27-30 in partially denaturing conditions generated an additional truncated fragment of about 16 kDa only in typical sporadic CJD (i.e. MM1). These results show that the physicochemical heterogeneity of PrP(Sc) in CJD extends to abnormal truncated forms of the protein. The findings support the notion of distinct structural "conformers" of PrP(Sc) and indicate that the characterization of truncated PrP(Sc) forms may further improve molecular typing in CJD.

The scrapie prion protein (PrP27-30) is a crucial component of the prion and is responsible for its transmissibility. Structural information on this protein is limited because it is insoluble and shows aggregated properties. In this study, PrP27-30 was effectively dispersed using sonication under the weak alkaline condition. Subsequently, the small PrP27-30 aggregates were subjected to different pH, heat, and denaturing conditions. The loss of proteinase K (PK) resistance of PrP27-30 and prion infectivity were monitored along with spectroscopic changes. Prion inactivation could not be achieved by the loss of PK resistance alone; a significant loss of the PrP27-30 amyloid structure, which was represented by a decrease in thioflavin T fluorescence, was required for the loss of transmissibility.

Extending the recent analysis of the safety of industrial bovine fat-derived products for human consumption (Müller, H., Stitz, L., and Riesner, D. (2006) Eur. J. Lip. Sci. Technol. 108, 812-826), we investigated systematically the effects of fat, fatty acids, and glycerol on the heat destruction of prions. Prion destruction was qualitatively and quantitatively evaluated in PrP 27-30, or prion rods, by the inactivation of infectivity as well as by the degradation of the polypeptide backbone. Under all conditions analyzed, inactivation of prion infectivity was achieved more efficiently than backbone degradation by several orders of magnitude. The presence of fat enhanced prion inactivation and offers a mild treatment for prion decontamination. In contrast, the presence of fat, fatty acids, and especially glycerol protected the PrP 27-30 backbone against heat-induced degradation. Glycerol also protected against heat-induced inactivation of prion infectivity. A phase distribution analysis demonstrated that prions migrated to the interphase of a fat/water mixture at room temperature and accumulated in the water phase at higher temperatures. In a systematic study of the mechanism of prion destruction, we found an intermediate structure of PrP that has fewer fibrils in beta-sheet formation, lower resistance to protease digestion, greater aggregation, and reduced solubility compared with PrP 27-30 but retains residual infectivity. These findings suggest that prion infectivity depends on beta-sheet-rich fibrillar structure and that inactivation proceeds in a stepwise manner, which explains the tailing effect frequently observed during inactivation.

The agent responsible for prion disease may exist in different forms, commonly referred to as strains, with each carrying the specific information that determines its own distinct biological properties, such as incubation period and lesion profile. Biological strain typing of ovine scrapie isolates by serial passage in conventional mice has shown some diversity in ovine prion strains. However, this biological diversity remains poorly supported by biochemical prion strain typing. The protein-only hypothesis predicts that variation between different prion strains in the same host is manifest in different conformations adopted by PrPSc. Here we have investigated the molecular properties of PrPSc associated with two principal Prnp(a) mouse-adapted ovine scrapie strains, namely, RML and ME7, in order to establish biochemical prion strain typing strategies that may subsequently be used to discriminate field cases of mouse-passaged ovine scrapie isolates. We used a conformation-dependent immunoassay and a conformational stability assay, together with Western blot analysis, to demonstrate that RML and ME7 PrPSc proteins show distinct biochemical and physicochemical properties. Although RML and ME7 PrPSc proteins showed similar resistance to proteolytic digestion, they differed in their glycoform profiles and levels of proteinase K (PK)-sensitive and PK-resistant isoforms. In addition, the PK-resistant core (PrP27-30) of ME7 was conformationally more stable following exposure to guanidine hydrochloride or Sarkosyl than was RML PrP27-30. Our data show that mouse-adapted ovine scrapie strains can be discriminated by their distinct conformers of PrPSc, which provides a basis to investigate their diversity at the molecular level.

An inert polysaccharide scaffold identified as a 5-15% component of prion rods (PrP 27-30) is unambiguously distinguishable from the N-glycosyl groups and the GPI anchor of PrP, and consists predominantly of 1,4-linked glucose with some branching via 1,4,6-linked glucose. We show that this polysaccharide scaffold is a common secondary component of prions found in hamster full-length PrP(Sc), prion rods and in mouse ScN2a prions from cell culture. The preparation from prion rods was improved, resulting in a polysaccharide scaffold free of remaining infectivity. Furthermore, we determined the stereochemistry of the glycoside linkages as pre-dominantly if not entirely alpha-glycosidic. The origin of the polysaccharide, its interaction with PrP and its potential relation to glycogen and corpora amylacea are discussed.

Elucidation of the structure of PrP(Sc) continues to be one of the most important and difficult challenges in prion research. This task, essential for gaining an understanding of the basis of prion infectivity, has been hampered by the insoluble, aggregated nature of this molecule. We used a combination of chemical cross-linking, proteolytic digestion, and mass spectrometry (MALDI-TOF and nanoLC-ESI-QqTOF), in an attempt to gain structural information about PrP 27-30 purified from the brains of Syrian hamsters infected with scrapie. The rationale of this approach is to identify pairs of specific amino acid residues that are close enough to each other to react with a bifunctional reagent of a given chain length. We cross-linked PrP 27-30 with the amino-specific reagent bis(sulfosuccinimidyl) suberate (BS(3)), obtaining dimers, trimers, and higher-order oligomers that were separated by SDS-PAGE. In-gel digestion followed by mass spectrometric analysis showed that BS(3) reacted preferentially with Gly90. A cross-link involving two Gly90 amino termini was found in cross-linked PrP 27-30 dimers, but not in intramolecularly cross-linked monomers or control samples. This observation indicates the spatial proximity of Gly90 amino termini in PrP 27-30 fibrils. The Gly90-Gly90 cross-link is consistent with a recent model of PrP 27-30, based on electron crystallographic data, featuring a fiber composed of stacked trimers of PrP monomers; specifically, it is compatible with cross-linking of monomers stacked vertically along the fiber axis but not those adjacent to each other horizontally in the trimeric building block. Our results constitute the first measured distance constraint in PrP(Sc).

For the surveillance of transmissible spongiform encephalopathies (TSEs) in animals and humans, the discrimination of different TSE strains causing scrapie, BSE, or Creutzfeldt-Jakob disease constitutes a substantial challenge. We addressed this problem by Fourier transform-infrared (FT-IR) spectroscopy of pathological prion protein PrP27-30. Different isolates of hamster-adapted scrapie (263K, 22A-H, and ME7-H) and BSE (BSE-H) were passaged in Syrian hamsters. Two of these agents, 22A-H and ME7-H, caused TSEs with indistinguishable clinical symptoms, neuropathological changes, and electrophoretic mobilities and glycosylation patterns of PrP27-30. However, FT-IR spectroscopy revealed that PrP27-30 of all four isolates featured different characteristics in the secondary structure, allowing a clear distinction between the passaged TSE agents. FT-IR analysis showed that phenotypic information is mirrored in beta-sheet and other secondary structure elements of PrP27-30, also in cases where immunobiochemical typing failed to detect structural differences. If the findings of this study hold true for nonexperimental TSEs in animals and humans, FT-IR characterization of PrP27-30 may provide a versatile tool for molecular strain typing without antibodies and without restrictions to specific TSEs or mammalian species.

The central event in the pathogenesis of prion diseases, a group of fatal, transmissible neurodegenerative disorders including Creutzfeldt-Jakob disease (CJD) in humans, is the conversion of the normal or cellular prion protein (PrPC) into the abnormal or scrapie isoform (PrPSc). The basis of the PrPC to PrPSc conversion is thought to involve the diminution of alpha-helical domains accompanied by the increase of beta structures within the PrP molecule. Consequently, treatment of PrPSc with proteinase K (PK) generates a large PK-resistant C-terminal core fragment termed PrP27-30 that in human prion diseases has a gel mobility of approximately 19-21 kDa for the unglycosylated form, and a ragged N terminus between residues 78 and 103. PrP27-30 is considered the pathogenic and infectious core of PrPSc. Here we report the identification of two novel PK-resistant, but much smaller C-terminal fragments of PrP (PrP-CTF 12/13) in brains of subjects with sporadic CJD. PrP-CTF 12/13, like PrP27-30, derive from both glycosylated as well as unglycosylated forms. The unglycosylated PrPCTF 12/13 migrate at 12 and 13 kDa and have the N terminus at residues 162/167 and 154/156, respectively. Therefore, PrP-CTF12/13 are 64-76 amino acids N-terminally shorter than PrP27-30 and are about half of the size of PrP27-30. PrP-CTF12/13 are likely to originate from a subpopulation of PrPSc distinct from that which generates PrP27-30. The finding of PrP-CTF12/13 in CJD brains widens the heterogeneity of the PK-resistant PrP fragments associated with prion diseases and may provide useful insights toward the understanding of the PrPSc structure and its formation.

It is now accepted that the structural transition from cellular prion protein (PrPC) to proteinase K-resistant prion protein scrapie (PrPSc) is the major event leading to transmissible spongiform encephalopathies. Although the mechanism of this transition remains elusive, glycosylation has been proposed to impede the PrPC to PrPSc conversion. To address the role of glycosylation, we have prepared glycosylated and unglycosylated peptides derived from the 175-195 fragment of the human prion protein. Comparison of the structure, aggregation kinetics, fibril formation capabilities, and redox susceptibility of Cys-179 has shown that the N-linked glycan (at Asn-181) significantly reduces the rate of fibrillization by promoting intermolecular disulfide formation via Cys-179. Further-more, the aggressive fibrillization of a C179S mutant of this fragment highlights the significant role of disulfide stability in retarding the rate of fibril formation. The implications of these studies are discussed in the context of fibril formation in the intact prion protein.

Prion diseases include Creutzfeldt-Jakob disease in humans, scrapie in sheep and bovine spongiform encephalopathy. The hallmark of prion diseases is the accumulation of an abnormal isoform (PrP(Sc)) of the cellular prion protein accompanied by neuronal cell death and astroglial proliferation. To characterize the correlation between PrP secondary and quarternary structure and their biological effects we assayed soluble and aggregated forms of PrP 27-30, the N-terminal truncated form of PrP(Sc), as well as the corresponding recombinant PrP(90-231) for their neurotoxicity and infectivity. PrP was kept soluble in 0.2% SDS and subsequently re-aggregated either by diluting the SDS or by adding acetonitril. The neurotoxicity of the re-aggregated states were comparable to that of prion rods (PrP 27-30) whereas the soluble forms had no neurotoxic effects. The solubilized PrP 27-30 showed no significant infection upon re-aggregation as determined by bioassays in Syrian golden hamsters. The recombinant PrP did not exhibit infectivity in any state.

The pathogenesis of prion (PrP) diseases is thought to be related to conformational changes of a normal cellular protein, PrPC, into a protease resistant protein called PrPSc, which is infectious by itself. A difficulty with this 'protein only' hypothesis is the existence of numerous PrP strains, that require PrPSc to have multiple conformations. Sporadic Creutzfeldt-Jakob disease (CJD), which accounts for nearly 80% of human prionoses, was reported to include at least two 'strains' termed types 1 and 2 which differ by electrophoretic patterns of their proteinase K (PK)-resistant fragments (PrP27-30). We have analyzed the biochemical and structural properties of PrPSc and PrP27-30 isolates from six sporadic CJD patients. Fourier transform-infra-red spectroscopy, PrP27-30 glycosylation patterns and studies of PK sensitivity revealed a striking heterogeneity. Furthermore, one isolate yielded a PrP27-30 fragment with a lower mobility clearly different from previously described sporadic CJD types. Although the average beta-sheet content was higher among type 1 isolates, there was overlap between the two types. Our study suggests that human sporadic CJD-related prions display a significant heterogeneity.

NMR has been used to refine the structure of Syrian hamster (SHa) prion protein rPrP(90-231), which is commensurate with the infectious protease-resistant core of the scrapie prion protein PrPSc. The structure of rPrP(90-231), refolded to resemble the normal cellular isoform PrPC spectroscopically and immunologically, has been studied using multidimensional NMR; initial results were published [James et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 10086-10091]. We now report refinement with better definition revealing important structural and dynamic features which can be related to biological observations pertinent to prion diseases. Structure refinement was based on 2778 unambiguously assigned nuclear Overhauser effect (NOE) connectivities, 297 ambiguous NOE restraints, and 63 scalar coupling constants (3JHNHa). The structure is represented by an ensemble of 25 best-scoring structures from 100 structures calculated using ARIA/X-PLOR and further refined with restrained molecular dynamics using the AMBER 4.1 force field with an explicit shell of water molecules. The rPrP(90-231) structure features a core domain (residues 125-228), with a backbone atomic root-mean-square deviation (RMSD) of 0.67 A, consisting of three alpha-helices (residues 144-154, 172-193, and 200-227) and two short antiparallel beta-strands (residues 129-131 and 161-163). The N-terminus (residues 90-119) is largely unstructured despite some sparse and weak medium-range NOEs implying the existence of bends or turns. The transition region between the core domain and flexible N-terminus, i.e., residues 113-128, consists of hydrophobic residues or glycines and does not adopt any regular secondary structure in aqueous solution. There are about 30 medium- and long-range NOEs within this hydrophobic cluster, so it clearly manifests structure. Multiple discrete conformations are evident, implying the possible existence of one or more metastable states, which may feature in conversion of PrPC to PrPSc. To obtain a more comprehensive picture of rPrP(90-231), dynamics have been studied using amide hydrogen-deuterium exchange and 15N NMR relaxation times (T1 and T2) and 15N{1H} NOE measurements. Comparison of the structure with previous reports suggests sequence-dependent features that may be reflected in a species barrier to prion disease transmission.

We have isolated RNA aptamers which are directed against the recombinant Syrian golden hamster prion protein rPrP23-231 (rPrPc) fused to glutathione S-transferase (GST). The aptamers did not recognize the fusion partner GST or the fusion protein GST::rPrP90-231 (rPrP27-30), which lacks 67 amino acids from the PrP N terminus. The aptamer-interacting region of PrPc was mapped to the N-terminal amino acids 23 to 52. Sequence analyses suggest that the RNA aptamers may fold into G-quartet-containing structural elements. Replacement of the G residues in the G quartet scaffold with uridine residues destroyed binding to PrP completely, strongly suggesting that the G quartet motif is essential for PrP recognition. Individual RNA aptamers interact specifically with prion protein in brain homogenates from wild-type mice (C57BL/6), hamsters (Syrian golden), and cattle as shown by supershifts obtained in the presence of anti-PrP antibodies. No interaction was observed with brain homogenates from PrP knockout mice (prn-p(0/0)). Specificity of the aptamer-PrP interaction was further confirmed by binding assays with antisense aptamer RNA or a mutant aptamer in which the guanosine residues in the G tetrad scaffold were replaced by uridine residues. The aptamers did not recognize PrP27-30 in brain homogenates from scrapie-infected mice. RNA aptamers may provide a first milestone in the development of a diagnostic assay for the detection of transmissible spongiform encephalopathies.

The scrapie prion protein (PrPSc) is formed from the cellular isoform (PrPC) by a post-translational process that involves a profound conformational change. Linear epitopes for recombinant antibody Fab fragments (Fabs) on PrPC and on the protease-resistant core of PrPSc, designated PrP 27-30, were identified using ELISA and immunoprecipitation. An epitope region at the C terminus was accessible in both PrPC and PrP 27-30; in contrast, epitopes towards the N-terminal region (residues 90 to 120) were accessible in PrPC but largely cryptic in PrP 27-30. Denaturation of PrP 27-30 exposed the epitopes of the N-terminal domain. We argue from our findings that the major conformational change underlying PrPSc formation occurs within the N-terminal segment of PrP 27-30., (Copyright 1997 Academic Press Limited.)

Prion diseases are neurodegenerative disorders characterized by the accumulation of abnormal isoforms of prion protein (PrPSc) in the central nervous system. PrPSc isoforms differ from their normal homologue (PrPC), in that they possess increased beta-sheet conformation, are partially protease resistant and may be associated with amyloid deposition. Amyloid proteins are thought to derive from soluble precursors or fragments thereof, present in biological fluids, which in the disease state undergo conformational change leading to aggregation and deposition in target tissues. We report here that platelets carry PrP mRNA and release PrPC, a sialoglycoprotein bound to the cell surface by a glycosylphosphatidylinositol (GPI) anchor. Soluble PrPC, and a N-terminal truncated PrPC isoform starting at position 90 are secreted by resting and agonist-stimulated platelets and are detectable after partial deglycosylation of releasates. N-terminal sequence analysis of the soluble 27-30 kDa isoform, GQGGGTHSQ(W)NKP, revealed homology to scrapie PrP27-30, the protease resistant core derived from PrPSc. These findings indicate that in addition to PrPC, platelets process a soluble PrP27-30 isoform. Whether this isoform can be converted in scrapie PrP27-30 remains to be determined.

The prion protein (PrP) undergoes a profound conformational change when the cellular isoform (PrPC) is converted into the scrapie form (PrPSc). Limited proteolysis of PrPsc produces PrP 27-30 which readily polymerizes into amyloid. To study the structure of PrP amyloid, we employed organic solvents that perturb protein conformation. Hexafluoro-2-propanol (HFIP), which promotes alpha-helix formation, modified the ultrastructure of rod-shaped PrP amyloids; flattened ribbons with a more regular substructure were found. As the concentration of HFIP was increased, the beta-sheet content and proteinase K resistance of PrP 27-30 as well as prion infectivity diminished. HFIP reversibly decreased the binding of Congo red dye to the rods while inactivation of prion infectivity was irreversible. In contrast to 10% HFIP, 1,1,1-trifluoro-2-propanol (TFIP) did not inactivate prion infectivity but like HFIP, TFIP did alter the morphology of the rods and abolish Congo red binding. This study separates prion infectivity from the amyloid properties of PrP 27-30 and underscores the dependence of prion infectivity on PrPSc conformation. The results also demonstrate that the specific beta-sheet-rich structures required for prion infectivity can be differentiated from those needed for amyloid formation as determined by Congo red binding.

The major, and possible only, component of the infectious prion is the scrapie prion protein (PrPSc); the protease resistant core of PrPSc is PrP 27-30, a protein of approximately 142 amino acids. PrPSc is derived from the cellular PrP isoform (PrPC) by a post-transliatonal process in which a profound conformational change occurs. Syrian hamster (SHa) PrP genes of varying length ranging from the N- and C- terminally truncated 90-228 up to the full-length mature protein 23-231 were inserted into various secretion and intracellular expression vectors that were transformed into Escherichia coli deficient for proteases. Maximum expression was obtained for a truncated SHaPrP containing residues 90-231, which correspond to the sequence of PrP 27-30; disruption of the bacteria using a microfluidizer produced the highest yields of this protein designated rPrP. After solubilization of rPrP in 8 M GdnHC1, it was purified by size exclusion chromatography and reversed phase chromatography. During purification the recovery was approximately 50%, and from each liter of E. coli culture, approximately 50 mg of purified rPrP was obtained. Expression of the longer species containing the basic N-terminal region was less successful and was not pursued further. The primary structure of rPrP was verified by Edman sequencing and mass spectrometry, and secondary structure determined by circular dichroism and Fourier transform infrared spectroscopy. When rPrP was purified under reducing conditions, it had a high beta-sheet content and relatively low solubility similar to PrPSc, particularly at pH values > 7. Refolding of rPrP by oxidation to form a disulfide bond between the two Cys residues of this polypeptide produced a soluble protein with a high alpha-helical content similar to PrPC. These multiple conformations of rPrP are reminiscent of the structural plurality that characterizes the naturally occurring PrP isoforms. The high levels of purified rPrP which can now be obtained should facilitate determination of the multiple tertiary structures that Prp can adopt.

PrP27-30 represents the protease-resistant core of the prion protein and was found to be the main component in Scrapie prion preparations. Recombinant (r) PrP27-30 corresponding to aa 90-231 from the Syrian golden hamster prion protein was expressed as a fusion with GST in E. coli and secreted from insect cells infected with recombinant baculoviruses, GST::rPrP27-30 isolated from either system was purified to homogenity by glutathione-Sepharose chromatography. rPrP27-30 from both systems was generated by direct cleavage of GST::rPrP27-30 in the presence of thrombin revealing a molecular weight of 17 kDa. GST::rPrP27-30 as well as the authentic protein rPrP27-30 were identified by immunoblotting employing a polyclonal antibody directed against a peptide corresponding to aa 95-110 of the Syrian golden hamster prion protein. In contrast to scrapie prior PrP27-30, the recombinant proteins GST::rPrP27-30 and rPrP27-30 were both sensitive towards proteinase K, suggesting that the molecules lack infectivity.

Certain neurodegenerative diseases in humans and animals are caused by small proteinaceous infectious particles called prions. Limited proteolysis and detergent extraction of the prions containing PrPSc generate prion rods that are composed of a polypeptide having an apparent molecular mass of 27 to 30 kDa. This polypeptide, termed prion protein PrP 27-30, has a ragged N terminus that begins at about residue 90, but retains scrapie infectivity. Moreover, the findings in a patient having an inherited prion disease of a truncated PrP with its C terminus at residue 145 suggest that the residues 90 to 145 may be of particular importance in the pathogenesis of prion diseases. To determine the three-dimensional organization of prion rods and to identify the core region involved in amyloid formation, we recorded X-ray diffraction patterns from rods purified from scrapie-infected Syrian hamster (SHa) brains which contain PrP 27-30, and from synthetic SHaPrP peptides. Three peptides were studied corresponding to residues 113 to 120 (peptide A8A, an octamer composed of glycines and alanines), 109 to 122 (H1, the first predicted alpha-helical region of PrPC), and 90 to 145 (a 56 residue peptide containing both H1 and the second predicted alpha-helical region of PrPC, H2). Electron microscopy, carried out in parallel with the X-ray measurements, revealed that all the samples formed linear polymers which were approximately 60 to approximately 200 A wide, with fibrillar or ribbon-like morphology. Gels and dried preparations of prion rods gave X-ray patterns that indicated a beta-sheet conformation, in which the hydrogen bond distance was 4.72 A and the intersheet distance was 8.82 A. For the three PrP peptides, the intersheet spacings varied widely, owing to the side-chains of the residues involved in the formation of the beta-sheet interactions, i.e., 5.13 A for A8A, 5.91 A for lyophilized H1, 7.99 A from solubilized and dried H1 and 9.15 A for the peptide SHa 90-145. The intersheet distance of PrP 27-30 was thus within the observed range for the peptides, and suggests that the amyloidogenic core of PrP is closely modeled by the peptide SHa 90-145.