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

The prion diseases seem to be caused by a conformational change of the prion protein (PrP) from the benign cellular form PrP C to the infectious scrapie form PrP Sc ; thus, detailed information about PrP structure may provide essential insights into the mechanism by which these diseases develop. In this study, the secondary structure of the recom-

The scrapie prion protein (PrP Sc ) is the major, and possibly the only, component of the infectious prion; it is generated from the cellular isoform (PrP C ) by a conformational change. N-terminal truncation of PrP Sc by limited proteolysis produces a protein of Ϸ142 residues designated PrP 27-30, which retains infectivity. A recombinant protein (rPrP) corresponding to Syrian hamster PrP 27-30 was expressed in Escherichia coli and purified. After refolding rPrP into an ␣-helical form resembling PrP C , the structure was solved by multidimensional heteronuclear NMR, revealing many structural features of rPrP that were not found in two shorter PrP fragments studied previously. Extensive side-chain interactions for residues 113-125 characterize a hydrophobic cluster, which packs against an irregular ␤-sheet, whereas residues 90-112 exhibit little defined structure. Although identifiable secondary structure is largely lacking in the N terminus of rPrP, paradoxically this N terminus increases the amount of secondary structure in the remainder of rPrP. The surface of a long helix (residues 200-227) and a structured loop (residues 165-171) form a discontinuous epitope for binding of a protein that facilitates PrP Sc formation. Polymorphic residues within this epitope seem to modulate susceptibility of sheep and humans to prion disease. Conformational heterogeneity of rPrP at the N terminus may be key to the transformation of PrP C into PrP Sc , whereas the discontinuous epitope near the C terminus controls this transition.

Prions are the transmissible pathogenic agents responsible for diseases such as scrapie and bovine spongiform encephalopathy. In the favoured model of prion replication, direct interaction between the pathogenic prion protein (PrPSc) template and endogenous cellular prion protein (PrPC) is proposed to drive the formation of nascent infectious prions. Reagents specifically binding either prion-protein conformer may interrupt prion production by inhibiting this interaction. We examined the ability of several recombinant antibody antigen-binding fragments (Fabs) to inhibit prion propagation in cultured mouse neuroblastoma cells (ScN2a) infected with PrPSc. Here we show that antibodies binding cell-surface PrPC inhibit PrPSc formation in a dose-dependent manner. In cells treated with the most potent antibody, Fab D18, prion replication is abolished and pre-existing PrPSc is rapidly cleared, suggesting that this antibody may cure established infection. The potent activity of Fab D18 is associated with its ability to better recognize the total population of PrPC molecules on the cell surface, and with the location of its epitope on PrPC. Our observations support the use of antibodies in the prevention and treatment of prion diseases and identify a region of PrPC for drug targeting.

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.

PrP(Sc) is known to be the major, if not the only, component of the infectious prion. Limited proteolysis of PrP(Sc) produces an N-terminally truncated polypeptide of about 142 residues, designated PrP 27-30. Recently, a recombinant protein (rPrP) of 142 residues corresponding to the Syrian hamster PrP 27-30 was expressed in Escherichia coli and purified (Mehlhorn et al., 1996). rPrP has been refolded into both alpha-helical and beta-sheet structures as well as various intermediates in aqueous buffers. The beta-sheet state and two pH-dependent alpha-helical states were characterized by CD and NMR. The alpha-helical conformation occurred only after the formation of an intramolecular disulfide bond, whereas the beta-sheet form was accessible either with or without the disulfide. Of the different alpha-helical forms studied, only those refolded in the pH range 5-8 were substantially soluble at physiological pH, exhibiting similar conformations and monomeric analytical sedimentation profiles throughout the above pH range. Furthermore, refolded alpha-rPrP showed NMR chemical shift dispersion typical of proteins with native conformations, although 2D NMR indicated large segments of conformational flexibility. It displayed a cooperative thermal denaturation transition; at elevated temperatures, it converted rapidly and irreversibly to the thermodynamically more stable beta-sheet form. Unfolding of alpha-rPrP by GdnHCl revealed a two-phase transition with a relatively stable folding intermediate at 2 M GdnHCl. The deltaG values were estimated to be 1.9 +/- 0.4 kcal/mol for the first phase and 6.5 +/- 1.2 kcal/mol for the second, consistent with a folding core surrounded by significant segments of flexible conformation. By NMR, alpha-rPrP(acid) isolated at pH 2 without refolding exhibited heterogeneous line widths, consistent with an acid-denatured molten globular state. We conclude that to the extent that rPrP constitutes a relevant folding domain of PrP(C), the various conformations exhibited by rPrP suggest that the PrP sequence may be intrinsically plastic in its conformations; indeed, portions of PrP(C) may possess a relatively open conformation which makes it susceptible to conversion into PrP(Sc) under appropriate conditions.

The prion diseases are an unusual group of fatal neurodegenerative disorders which include Creutzfeldt-Jakob disease, fatal familial insomnia, and Gerstmann-Sträussler-Scheinker disease in humans as well as bovine spongiform encephalopathy in cattle and scrapie in sheep and goats. A wealth of evidence supports the hypothesis that these diseases are caused by a conformational change in the prion protein (PrP) from its normal, cellular isoform (PrP C ) into a pathogenic, infectious isoform (PrP Sc ) (17,18,21). To investigate the pathogenesis of prion diseases and to identify potential therapeutic targets, much effort is now directed toward characterizing the structural biology of PrP Sc formation. Recent nuclear magnetic resonance studies of recombinant PrP molecules provide evidence for a three-␣-helix bundle protein with a short ␤-strand region and a relatively unstructured N terminus (4,7,9,23,24). These structural data have facilitated the rational analysis of mutagenesis-specific PrP segments in order to determine their importance for prion propagation.As part of a systematic PrP deletion mutagenesis study, we discovered that Tg(MHM2,⌬23-88)9381/Prnp 0/0 mice expressing N-terminally truncated MHM2 PrP molecules were resistant to Rocky Mountain Laboratory (RML) murine prions (30). MHM2 is a chimeric construct that differs from wild-type mouse (Mo) PrP at positions 108 and 111 (28). Substitution at these positions with the homologous residues from the Syrian hamster (SHa) PrP sequence (L108M and V111M) creates an epitope for the anti-PrP 3F4 monoclonal antibody (MAb) (8). Although Tg(MHM2,⌬23-88)9381/Prnp 0/0 mice failed to propagate RML prions, MHM2(⌬23-88) molecules expressed in scrapie-infected mouse neuroblastoma (ScN2a) cells successfully formed protease-resistant MHM2(⌬23-88)PrP Sc (13). Furthermore, Tg(MHM2,⌬23-88)9381/Prnp ϩ/0 mice expressing endogenous MoPrP in addition to the truncated, chimeric transgene were susceptible to RML prion infection. These heterozygote mice developed scrapie ϳ260 days after inoculation with RML prions, and biochemical analysis revealed the accumulation of protease-resistant MHM2(⌬23-88)PrP Sc in their brains (30). These complementary results in ScN2a cells and Prnp ϩ/0 mice indicate that coexpression of MoPrP facilitates the conversion of MHM2(⌬23-88)PrP C to MHM2(⌬23-88)PrP Sc . In view of the foregoing results, it remained unclear why Tg(MHM2,⌬23-88)9381/Prnp 0/0 mice were not susceptible to RML prions in the absence of MoPrP. Immunofluorescence studies did not reveal any significant differences in the cellular localization of truncated, chimeric, and wild-type PrP molecules in transfected mouse neuroblastoma (N2a) cells (data not shown). Possible explanations for the resistance of Tg-(MHM2,⌬23-88)9381/Prnp 0/0 mice to prion infection include (i) a prion transmission barrier between full-length mouse PrP and MHM2(⌬23-88), (ii) decreased prion conversion efficiency caused by removal of the N terminus, (iii) insufficient expression of the MHM2(⌬23-88) transgene, and (iv) ...

The N-terminally truncated form of the prion protein, PrP 27-30, and the corresponding recombinant protein, rPrP, were solubilized in 0.2% SDS, and the transitions induced by changing the conditions from 0.2% SDS to physiological conditions, i.e. removing SDS, were characterized with respect to solubility, resistance to proteolysis, secondary structure and multimerization. Circular dichroism, electron microscopy and fluorescence correlation spectroscopy were used to study the structural transitions of PrP. Within one minute the alpha-helical structure of PrP was transformed into one that was enriched in beta-sheets and consisted mainly of dimers. Larger oligomers were found after 20 minutes and larger multimers exhibiting resistance to proteolysis were found after several hours. It was concluded that the monomeric alpha-helical conformation was stable in SDS or when attached to the membrane; however, the state of lowest free energy in aqueous solution at neutral pH seems to be the multimeric, beta-sheet enriched conformation.