Ziwei Huang, Stanley B Prusiner and Fred E Cohen Folding & Design 1996, 1: 13-19.
A conformational change seems to represent the major difference between the scrapie prion protein (PrPSc) and its normal cellular isoform (PrPC). We recently proposed a set of four helix bundle models for the three-dimensional structure of PrPC that are consistent with a variety of spectroscopic and genetic data. We report a plausible model for the three-dimensional structure of a biologically important fragment of PrPSc. The model of residues 108-218 was constructed by an approach that combines computational techniques and experimental data. The proposed structures of this fragment of PrPSc display a four-stranded ■-sheet covered on one face by two -helices. Residues implicated in the prion species barrier are found to cluster on the solvent-accessible surface of the ■-sheet of one of the models. This interface could provide a structural template that would assist the conversion of PrPC to PrPSc and hence direct prion propagation.Molecular models of the PrP isoforms should prove very useful in developing structural hypotheses about the process by which PrPC is transformed into PrPSc, the mechanisms by which PrP gene mutations give rise to the inherited human prion diseases, and the species barrier that seems to protect humans from animal prions. It seems likely that PrPC represents a kinetically trapped intermediate in PrP folding. Introduction and background Prions cause a group of human and animal neurodegenerative diseases that are now classified together because their etiology and pathogenesis involve modification of the prion protein (PrP)  . Prion diseases are manifest as infectious, genetic and sporadic disorders. These diseases can be transmitted among mammals by the infectious particle designated 'prion' . Despite intensive searches over the past three decades, no nucleic acid has been found within prions    ; yet, a modified isoform of the host-encoded PrP, designated PrPSc, is essential for infectivity     . In fact, considerable experimental data argue that prions are composed exclusively of PrPSc. Earlier terms used to describe the prion diseases include transmissible encephalopathies, spongiform encephalopathies and slow virus diseases   . The quartet of human prion diseases are frequently referred to as kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Ströussler-Scheinker (GSS) disease and fatal familial insomnia (FFI). Kuru was the first of the human prion diseases to be transmitted to experimental animals and it has often been suggested that kuru spread among the Fore people of Papua New Guinea by ritualistic cannibalism  . The experimental and presumed human to human transmission of kuru led to the belief that prion diseases are infectious disorders caused by unusual viruses similar to those causing scrapie in sheep and goats. Yet, a paradox was presented by the occurrence of CJD in families, first reported almost 70 years ago  , which appeared to be a genetic disease. The significance of familial CJD was unappreciated until mutations were discovered in the protein-coding region of the PrP gene on the short arm of chromosome 20   . The earlier finding that brain extracts from patients who had died of familial prion diseases inoculated into experimental animals often transmit disease posed a conundrum that was resolved with the genetic linkage of these diseases to mutations of the PrP gene   . The most common form of prion disease in humans is sporadic CJD. Many attempts to show that the sporadic prion diseases are caused by infection have been unsuccessful    . The discovery that inherited prion diseases are caused by germline mutation of the PrP gene raised the possibility that sporadic forms of these diseases might result from a somatic mutation . The discovery that PrPSc is formed from the cellular isoform of the prion protein, PrPC, by a posttranslational process  and that overexpression of wildtype PrP transgenes produces spongiform degeneration and infectivity de novo  has raised the possibility that sporadic prion diseases result from the spontaneous conversion of PrPC into PrPSc. The fundamental event in prion diseases seems to be a conformational change in PrP . All attempts to identify a posttranslational chemical modification that distinguishes PrPSc from PrPC have been unsuccessful to date . PrPC contains about 45% -helix and is virtually devoid of ■-sheet . Conversion to PrPSc creates a protein that contains about 30% -helix and about 45% ■-sheet. The mechanism by which PrPC is converted into PrPSc remains unknown but PrPC appears to bind to PrPSc to form an intermediate complex during the formation of nascent PrPSc. Transgenic mouse studies have demonstrated that PrPSc in the inoculum interacts preferentially with homotypic PrPC during the propagation of prions  . Elucidating the three-dimensional structure of both PrPC and PrPSc is central to understanding the molecular mechanism of prion diseases. An approximate tertiary structure of PrPC was proposed recently that is rich in -helical structures consistent with available spectroscopic data . As the insoluble aggregated nature of PrPSc complicates X-ray and NMR studies of its three-dimensional structure, we have utilized an approach that combines computational techniques and a synthesis of available data from genetic, molecular biological, and spectroscopic experiments. Our approach includes secondary structure prediction, a combinatorial search of all plausible arrangements of these secondary structure elements to form tertiary structures, and an analysis of the putative tertiary structures to identify those candidates that are consistent with a variety of pieces of experimental and theoretical information. In an effort to simplify this task, we chose to focus on a biologically relevant fragment of PrPSc. PrP includes three regions: an N-terminal leader sequence (residues 1-23); the octarepeats, a sequentially regular region with a pattern of amino acid side chains that is not easily related to other known periodic structures (residues 24-90); and the 27-30kDa infectious fragment, PrP 27-30, that remains after proteinase K digestion of PrPSc (residues 90-240), which includes two potential glycosylation sites and a glycophosphoinositol anchor near the C terminus. Although polymorphisms in the octarepeat region have been associated with inherited disease, a comparison of the octarepeat regions of primate PrP sequences demonstrate that these are normal polymorphisms . We have chosen to focus on the third region because of the connection to infectivity, the fact that the vast majority of disease-associated mutations and disease-modifying polymorphisms occur in this region, and because of the demonstration that peptides chosen from this region (e.g. 90-145 and 108-141) undergo an -helix to ■-structure transition in vitro that is reminiscent of the conversion of PrPC into PrPSc . The fact that a Japanese patient with a missense mutation at codon 145 developed a neurodegenerative illness argues that the crux of the conformational change must occur between residues 90 and 145 . Results and discussion The secondary structure of PrPSc was predicted with specific biases introduced to accommodate data from circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopic experiments. CD and FTIR studies indicate that approximately half of the -helical structure in PrPC is converted into ■-sheet in PrPSc  . Results from various secondary structure prediction methods and spectroscopic studies of PrP peptide fragments argue that the four structural regions corresponding to the helices in PrPC are most likely to undergo a conformational change from -helix to ■-sheet in PrPSc  . These findings suggest that two of the four helices of PrPC are converted into ■-sheet structure in PrPSc. We therefore systematically assigned two of the four putative helical regions to ■-sheet conformations. The tertiary structures of PrPSc were generated by examining the plausible hydrophobic packing between secondary structures. A combinatorial packing approach developed for /■ proteins   was employed to predict tertiary structural models for PrPSc. All possible strand arrangements and relative pairing of the component strands to form a ■-sheet were studied. Briefly, each of the four strands could occupy one of 4! orders (1234, 2143, etc.) and there are two possible orientations for each ■-strand. Strand alignments are shifted to create four distinct hydrogen bonding patterns for each strand. Thus, a large number of structures are generated with unique strand topologies and hydrogen bonding patterns, including parallel, antiparallel, and mixed ■-sheets. The separation between the axes of the ■-strands was 4.25 ë to facilitate hydrogen bonding and the angle between neighboring strands was -20â to create the twisted ■-sheet that is commonly observed in protein structures . The -helices were then placed onto the hydrophobic surface of the ■-sheet structure so that a constellation of non-polar residues on the surface of each -helix could interact with the hydrophobic surface of the ■-sheet. Helices were placed antiparallel to the preceding secondary structure element 10 ë above the plane of the sheet. This organization is typical of /■ and +■ protein structures. Structures were eliminated if their ■-sheets failed to create a hydrophobic surface suitable for subsequent helix-sheet packing or if the ■-sheet topology created steric problems for the loops that would ultimately join the ■-strands. For example, if two six-residue strands were joined by a four-residue turn, they could not run parallel to one another in a ■-sheet without breaking the chain. Structures were also eliminated if they failed to form the experimentally determined disulfide bond between Cys179 and Cys214 . From an initial list of about 106 structures, six families of structures were considered viable [Fig. 1]. These six structural models were validated by a three-dimensional profile method . The profile scores are presented in [Table 1]. Although self-profiling methods cannot guarantee the accuracy of a given model structure, they are useful in identifying errant folds and alignments. Table 1. Three-dimensional profiles for the six models of PrPSc Model Score Expected score range I 11.54 13.96-31.03 II 18.63 13.96-31.03 III 14.39 13.96-31.03 IV 11.82 12.94-28.76 V 10.11 12.94-28.76 VI 16.12 12.94-28.76 Fig. 1. Schematic drawings of the six plausible structures of PrPSc. These structural models were selected from an initial list of about 106 structures. ■-strands are represented by an arrow and -helices by a rectangle. Helices are numbered A2 (129-141), A3 (178-191) and A4 (202-218), and strands are labeled S1a, S1b, S2a, S2b, S4a, and S4b. See text for strand N and C termini.
The generated three-dimensional model structures were examined in order to select the models that were most consistent with experimental data. Genetic experiments indicated that a specific interaction between PrPSc and PrPC is important for prion propagation  . A small number of residues were found to be central to the prion species barrier, a phenomenon that depends upon the homotypic interaction of PrPSc with PrPC . Each of the six plausible structures [Fig. 1] was analyzed with respect to the residues implicated in the PrPSc-PrPC interaction (Asn108, Met112, Met129, and Ala133). One of the models was most intriguing with respect to the spatial clustering of these residues. As shown in [Fig. 2a], this model displays a four-stranded ■-sheet with one face covered by two -helices. The ■-strand arrangement in the ■-sheet is similar to that observed in the structure of the immunoglobulin-binding domain of protein G (GB1) , and the overall packing topology between the ■-sheet and two -helices is similar to that observed in the structure of the RNA-binding domain of the human heterogeneous nuclear ribonucleoprotein (hnRNP) C protein . The four residues implicated in the species barrier, and thus the PrPSc-PrPC interaction, are clustered on the solvent-accessible surface of the ■-sheet of model II. In addition, a number of hydrophobic side chains are located on this surface. These observations suggest that the solvent-accessible surface of the ■-sheet provides a plausible interface for a specific interaction of PrPSc with PrPC that could promote the conformational change of PrPC to PrPSc. Mutations at this interface would alter the incubation times associated with different inocula of prions in different hosts. This suggests that the prion species barrier results from the changes in the conformation and stability of the PrPSc-PrPC complex. Transgenic experiments are underway to create new and abrogate existing species barriers based on this model. Although we currently favor model II of PrPSc, it is important to remember that each model is emblematic of a family of protein topologies, and that formal experimental or theoretical constraints prevent one from excluding the other five plausible models. The model of the PrPSc-PrPC complex may provide a structural basis for observations from genetic experiments. Transgenic studies of the transmission of prions from Syrian hamster to mouse indicate that five residues (109, 112, 138, 154, and 169) are important for the homophilic interaction between PrPSc molecules within the inoculated prion and PrPC synthesized by the host . It is interesting to note that three of these five residues (109, 112, and 138) are located at or near the hypothesized PrPSc-PrPC interface [Fig. 2a], while the remaining two residues (154 and 169) are located in the S2b-H3 loop connecting the fourth strand in the ■-sheet and helix 3. This suggests that, in addition to the ■-sheet being the primary site for interaction, the loop following the ■-sheet might also be involved in molecular recognition during prion replication. This would be consistent with model II for PrPSc, in which this conformationally flexible loop could come into contact with PrPC during the formation of the PrPSc-PrPC complex [Fig. 2a]. Such a model also provides a consistent explanation for the recent studies of the transmission of CJD from humans to transgenic mice. Mice expressing a chimeric PrP gene containing nine substitutions were susceptible to prions from CJD patients . Eight of these nine substitutions are located either in the putative ■-sheet region of model II or the S2b-H3 loop region of the PrPSc model. Analyses of PrP genes among various primates indicated that mutations at residue 177 and a polymorphism at residue 168 are implicated in the prion species barrier  . These findings provide further evidence for the possible structural or functional role of the putative S2b-H3 loop. Fig. 2. Plausible models for the tertiary structures of PrPSc and PrPC. (a) The proposed three-dimensional structure of PrPSc. This structure was chosen from the six penultimate models of PrPSc [Fig. 1] because it appeared to correlate best with genetic data on residues involved in species barrier. It contains a four-strand mixed ■-sheet with two -helices packed against one face of the ■-sheet. Strands 1a and 1b (in red) correspond to the helix 1 in PrPC while strands 2a and 2b (in green) correspond to the helix 2 (b). Helices 3 and 4 in this model remain unchanged from the PrPC model (b) . Four residues (Asn108, Met112, Met129, and Ala133) implicated in the species barrier  are shown in the ball- and-stick model. They cluster on the solvent-accessible surface of the ■-sheet, which might provide a plausible interface for the PrPSc-PrPC interaction. The S2b-H3 loop connecting the ■-sheet and helix 3 is implicated in the species barrier and is shown in yellow. This conformationally flexible loop could come into contact with PrPC during the formation of the PrPSc-PrPC complex. Therefore, the specific molecular recognition during prion replication might involve both the ■-sheet as the primary binding site and the S2b-H3 loop as an additional site for interaction. (b) The proposed three-dimensional structure of PrPC . Helix 1 is shown in red and helix 2 in green. We believe that helices 1 and 2 are converted into ■-sheet structure during the formation of PrPSc (a). The H2-H3 loop corresponding to the S2b-H3 loop in PrPSc is shown in yellow. Four residues (Asn108, Met112, Met129, and Ala133) implicated in the species barrier as noted above are shown in the ball-and-stick model. Recently, other authors raised the issue that overall sequence homology might not be a determining influence for prion transmissibility . This hypothesis was based on the finding that, despite the sequence similarity to humans, Old World rhesus monkeys exhibit a lower transmission rate of human prions than either of the more evolutionarily distal New World spider or squirrel monkeys. Examination of these results in the context of the proposed model for the PrPSc-PrPC interaction reveals that PrP from Old World rhesus monkey contains a substitution at codon 108 (AsnSer) at the solvent-accessible surface of the putative ■-sheet, a primary site of PrPSc-PrPC interface [Fig. 2a]. This substitution might decrease the stability of the PrPSc-PrPC complex and hence lower the transmission rate of human prions. Thus, our structural model for prion replication suggests that the sequence, or more precisely structural similarity, at the PrPSc-PrPC interface, rather than the overall sequence homology, may be the major determinant for prion transmissibility. The proposed structural model of PrPSc is consistent with the mutation data in familial prion diseases. 18 mutations in the PrP gene have been identified that segregate with the inherited prion diseases  . Although octarepeat inserts at codons 67, 75, or 83 of the N-terminal region are either genetically linked to or associated with familial CJD, all other disease-associated point mutations occur in the C-terminal region, in which the ■-sheet and -helical structures are predicted to occur. As the deletion of the N-terminal region of PrPSc before residue 90 by limited proteolysis does not alter prion infectivity, this suggests that the N-terminal region is not required for the propagation of prions. Point mutations in the C-terminal region may affect the structure of either PrPC or PrPSc and promote the conformational change featured in prion diseases. It was found, for example, that 10 mutations are located within or near the four putative -helices and five mutations cluster around a central hydrophobic core that seems essential for the structural stability of PrPC [Fig. 2b] . It is noted that a nonsense mutation at codon 145 was found in a Japanese patient dying of CJD . This finding suggests that residues 90-145 might be essential for the transmission and pathology of prion diseases. This would be consistent with the proposed model of PrPSc in which residues 90-145 form the region involved in the conformational change of two -helices into a four-stranded ■-sheet. This same region contains the clustering of residues implicated in the species barrier for prion transmission [Fig. 2a,b]. The proposed structure of PrPSc is also consistent with results from structural studies of PrP peptides. Spectroscopic studies have shown that the synthetic peptide corresponding to residues 109-122 (■-strands 1a and 1b) or 128-141 (■-strands 2a and 2b) of model II of PrPSc adopts a ■-sheet structure and forms amyloid    . Further structural studies using NMR, FTIR and CD spectroscopy of peptides containing both the first and second putative hairpin structures (e.g. residues 90-145) suggest that this region can adopt two distinct conformations: one that contains a pair of helices and a second that is entirely ■-structure . All of these findings seem to be in agreement with the structural features revealed in the models of PrPSc and PrPC [Fig. 2] and of the formation of a complex between PrPC and PrPSc which seems to occur during prion propagation. Conclusions The models for PrPSc proposed here extend earlier studies in which computational models were developed for PrPC . Both the PrPC and PrPSc models were developed while considering numerous constraints posed by the physical, genetic and amino acid sequence data available for these two PrP isoforms. These models should prove very useful in posing hypotheses about the process by which PrPC is transformed into PrPSc, the mechanisms by which PrP gene mutations give rise to the inherited human prion diseases, and the species barrier that seems to protect humans from animal prions. In the absence of more definitive structural information, we believe that these models provide a useful platform for designing genetic and biophysical experiments to probe the unique biology of prion diseases. Materials and methods The computational procedures used for the prediction of the three-dimensional structures of PrPSc involved four major steps: (i) alignment of a family of homologous sequences, (ii) prediction of secondary structures, (iii) packing of secondary elements to generate all plausible tertiary structures, and (iv) selection and refinement of final structural models. PrP amino acid sequences from one avian and 11 mammalian sources, including chicken, cow, sheep, rat, mouse, hamster, mink, and human, were used. The alignment of these sequences was reported previously . Methods for secondary and tertiary structure prediction were applied independently to all 12 PrP sequences using the statistical methods of Chou and Fasman  and Garnier et al. ; the neural networks of Kneller et al.  and Rost and Sander ; and the pattern-based method of Cohen et al. . These methods gave rise to a consistent prediction of secondary structure location, but a confusing picture of the local preference for -helical or ■-sheet structure. We systematically assigned two of the four helical regions in PrPC [Fig. 2b]  to ■-sheet conformations and thus obtained six different sets of secondary structure for PrPSc, i.e. S1a1bS2a2bH3H4, S1a1bH2S3a3bH4, S1a1bH2H3S4a4b, H1S2a2bS3a3bH4, H1S2a2bH3S4a4b, and H1H2S3a3bS4a4b. The boundaries of the ■-strands and the ■-turns of the hairpins were predicted using a turn-prediction algorithm for /■ proteins . The predicted boundaries of the plausible ■-strands in PrPSc are Asn108-Ala113 for S1a, Ala116-Val122 for S1b, Tyr128-Ser135 for S2a, Ile138-Asp144 for S2b, Asp178-Ile184 for S3a, His187-Thr191 for S3b, Asp202-Val210 for S4a, and Met213-Tyr218 for S4b. The predicted boundaries of the -helices in PrPSc remain unchanged from the PrPC model (Met109-Val122 for H1, Met129-Phe141 for H2, Asp178-Thr191 for H3, and Asp202-Tyr218 for H4) . Thus, the models attempt to describe the structure for the 111-residue PrP fragment from residue 108 to residue 218. The predicted secondary structure corresponds to 64% of the fragment for which 36% is ■-structure (24% intramolecular hydrogen bonds within a PrPSc monomer and about 12% intermolecular hydrogen bonds between ■-strands in PrPSc monomers that form a multimeric state) and 28% is -helical. These figures modestly underestimate the values expected from the FTIR studies and could easily reach the experimental values if the extent of the intermolecular hydrogen bonding is larger. The tertiary structures of PrPSc were generated by examining the plausible hydrophobic packing between secondary structure elements . Specifically, we sought ■-sheets that created an appropriate hydrophobic surface to support helix-sheet packing and identified hydrophobic clusters on the surfaces of putative -helices to interact with the hydrophobic regions of the ■-sheet. Finally, a series of experimental and theoretical constraints were applied to select plausible structural models. For example, the existence of a disulfide bridge between residues 179 and 214 constrains the separation of the predicted locations for the and ■ carbons for these two residues. Also, the separation between the end of one piece of secondary structure and the beginning of the next should not exceed a length that could be spanned by the intervening residues.
References 1.Prusiner S.B : (1991). Molecular biology of prion diseases. Science (1991). 252 : 1515-1522. 2.Prusiner S.B. : (1995). The prion diseases. Sci. Am. 272 : 48-57. 3.Prusiner S.B. : (1982). Novel proteinaceous infectious particles cause scrapie. Science 216 : 136-144. 4.Alper T. , Haig D.A. , Clarke M.C. : (1966). The exceptionally small size of the scrapie agent. Biochem. Biophys. Res. Commun. 22 : 278-284. 5.Alper T. , Cramp W.A. , Haig D.A. , Clarke M.C. : (1967). Does the agent of scrapie replicate without nucleic acid? Nature 214 : 764-766. 6.Hunter G.D. : (1972). Scrapie: a prototype slow infection. J. Infect. Dis. 125 : 427-440. 7.Riesner D. , Kellings K. , Meyer N. , Mirenda C. , Prusiner S.B. : (1992). Nucleic acids and scrapie prions. In Prion Diseases of Humans and Animals. (Prusiner, S.B., Collinge, J., Powell, J. & Anderton, B.,, eds.) 341-358. Ellis Horwood, London. 8.Prusiner S.B. et al. , Glenner G.G. : (1983). Scrapie prions aggregate to form amyloid-like birefringent rods. Cell 35 : 349-358. 9.BŞeler H. et al. , Weissmann C. : (1993). Mice devoid of PrP are resistant to scrapie. Cell 73 : 1339-1347. 10.Prusiner S.B. et al. , DeArmond S.J. : (1993). Ablation of the prion protein (PrP) gene in mice prevents scrapie and facilitates production of anti-PrP antibodies. Proc. Natl. Acad. Sci. USA 90 : 10608-10612. 11.Prusiner S.B. , Groth D. , Serban A. , Stahl N. , Gabizon R. : (1993). Attempts to restore scrapie prion infectivity after exposure to protein denaturants. Proc. Natl. Acad. Sci. USA 90 : 2793-2797. 12.Sigurdsson B. : (1954). Rida, a chronic encephalitis of sheep with general remarks on infections which develop slowly and some of their special characteristics. Br. Vet. J. 110 : 341-354. 13.Gajdusek D.C. : (1977). Unconventional viruses and the origin and disappearance of kuru. Science 197 : 943-960. 14.Gajdusek D.C. : (1985). Subacute spongiform virus encephalopathies caused by unconventional viruses. In Subviral Pathogens of Plants and Animals: Viroids and Prions. (Maramorosch, K. & McKelvey, J.J., Jr., eds.) Academic Press, Orlando. 483-544. 15.Gajdusek D.C. , Gibbs C.J.Jr. , Alpers M. : (1966). Experimental transmission of a kuru-like syndrome to chimpanzees. Nature 209 : 794-796. 16.Kirschbaum W.R. : (1924). Zwei eigenartige Erkrankungen des Zentralnervensystems nach Art der spastischen Pseudosklerose (Jakob). Z. Ges. Neurol. Psychiatr. 92 : 175-220. 17.Meggendorfer F. : (1930). Klinische und genealogische Beobachtungen bei einem Fall von spastischer Pseudosklerose Jakobs. Z. Ges. Neurol. Psychiatr. 128 : 337-341. 18.Sparkes R.S. et al. , Weiner L.P. : (1986). Assignment of the human and mouse prion protein genes to homologous chromosomes. Proc. Natl. Acad. Sci. USA 83 : 7358-7362. 19.Hsiao K. et al. , Prusiner S.B. : (1989). Linkage of a prion protein missense variant to Gerstmann-Ströussler syndrome. Nature 338 : 342-345. 20.Prusiner S.B. : (1994). Inherited prion diseases. Proc. Natl. Acad. Sci. USA 91 : 4611-4614. 21.Masters C.L. , Gajdusek D.C. , Gibbs C.J.Jr. : (1981). Creutzfeldt-Jakob disease virus isolations from the Gerstmann-Ströussler syndrome. Brain 104 : 559-588. 22.Prusiner S.B. : (1989). Scrapie prions. Annu. Rev. Microbiol. 43 : 345-374. 23.Tateishi J. et al. , Boellaard J.W. : (1992). Prion protein gene analysis and transmission studies of Creutzfeldt-Jakob disease. In Prion Diseases of Humans and Animals. (Prusiner, S.B., Collinge, J., Powell, J. & Anderton, B., eds.) 129-134. Ellis Horwood, London. 24.Malmgren R. , Kurland L. , Mokri B. , Kurtzke J. : (1979). The epidemiology of Creutzfeldt-Jakob disease. In Slow Transmissible Diseases of the Nervous System, vol 3. (Prusiner, S.B. & Hadlow, W.J.,, eds.) 93-112. Academic Press, New York. 25.Brown P. , Cathala F. , Raubertas R.F. , Gajdusek D.C. , Castaigne P. : (1987). The epidemiology of Creutzfeldt-Jakob disease: conclusion of 15-year investigation in France and review of the world literature. Neurology 37 : 895-904. 26.Harries-Jones R. , Knight R. , Will R.G. , Cousens S. , Smith P.G. , Matthews W.B. : (1988). Creutzfeldt-Jakob disease in England and Wales, 1980-1984: a case-control study of potential risk factors. J. Neurol. Neurosurg. Psychiatry 51 : 1113-1119. 27.Cousens S.N. , Harries-Jones, R. , Knight R. , Will R.G. , Smith P.G. , Matthews W.B. : (1990). Geographical distribution of cases of Creutzfeldt-Jakob disease in England and Wales 1970-84. J. Neurol. Neurosurg. Psychiatry 53 : 459-465. 28.Borchelt D.R. , Scott M. , Taraboulos A. , Stahl N. , Prusiner S.B. : (1990). Scrapie and cellular prion proteins differ in their kinetics of synthesis and topology in cultured cells. J. Cell Biol. 110 : 743-752. 29.Westaway D. et al. , Prusiner S.B. : (1994). Degeneration of skeletal muscle, peripheral nerves, and the central nervous system in transgenic mice overexpressing wild-type prion proteins. Cell 76 : 117-129. 30.Cohen F.E. , Pan K.-M. , Huang Z. , Baldwin M. , Fletterick R.J. , Prusiner S.B. : (1994). Structural clues to prion replication. Science 264 : 530-531. 31.Stahl N. et al. , Prusiner S.B. : (1993). Structural analysis of the scrapie prion protein using mass spectrometry and amino acid sequencing. Biochemistry 32 : 1991-2002. 32.Pan K.-M. et al. , Prusiner S.B. : (1993). Conversion of -helices into ■-sheets features in the formation of the scrapie prion proteins. Proc. Natl. Acad. Sci. USA 90 : 10962-10966. 33.Prusiner S.B. et al. , DeArmond S.J. : (1990). Transgenetic studies implicate interactions between homologous PrP isoforms in scrapie prion replication. Cell 63 : 673-686. 34.Scott M. et al. , Prusiner S.B. : (1993). Propagation of prions with artificial properties in transgenic mice expressing chimeric PrP genes. Cell 73 : 979-988. 3; 35.Huang Z. , Gabriel J.-M. , Baldwin M.A. , Fletterick R.J. , Prusiner S.B. , Cohen F.E. : (1994). Proposed three-dimensional structure for the cellular prion protein. Proc. Natl. Acad. Sci. USA 91 : 7139-7143. 3; 4; 5; 6; 7; 8; 36.Schötzl H.M. , Da Costa M. , Taylor L. , Cohen F.E. , Prusiner S.B. : (1995). Prion protein gene variation among primates. J. Mol. Biol. 245 : 362-374. 3; 4; 37.Zhang H. et al. , Prusiner S.B. : (1995). Conformational transitions in peptides containing two putative -helices of the prion protein. J. Mol. Biol. 250 : 514-526. 38.Kitamoto T. , Ohta M. , Doh-ura K. , Hitoshi S. , Terao Y. , Tateishi J. : (1993). Novel missense variants of prion protein in Creutzfeldt-Jakob disease or Gerstmann-Ströussler syndrome. Biochem. Biophys. Res. Commun. 191 : 709-714. 39.Safar J. , Roller P.P. , Gajdusek D.C. , Gibbs C.J.Jr. : (1993). Conformational transitions, dissociation, and unfolding of scrapie amyloid (prion) protein. J. Biol. Chem. 268 : 20276-20284. 40.Gasset M. et al. , Prusiner S.B. : (1992). Predicted -helical regions of the prion protein when synthesized as peptides form amyloid. Proc. Natl. Acad. Sci. USA 89 : 10940-10944. 41.Cohen F.E. , Sternberg M.J.E. , Taylor W.R. : (1982). The analysis and prediction of the tertiary structure and globular proteins involving the packing of -helices against a ■-sheet: a combinatorial approach. J. Mol. Biol. 156 : 821-862. 3; 42.Hurle M.R. , Matthews C.R. , Cohen F.E. , Kuntz I.D. , Toumadje A. , Johnson C.W. : (1987). Prediction of the tertiary structure of the -subunit of tryptophan synthase. Proteins: Structure Function and Genetics 2 : 210-224. 43.Turk E. , Teplow D.B. , Hood L.E. , Prusiner S.B. : (1988). Purification and properties of the cellular and scrapie hamster prion proteins. Eur. J. Biochem. 176 : 21-30. 44.Luthy R. , Bowie J.U. , Eisenberg D. : (1992). Assessment of protein models with three-dimensional profiles. Nature 356 : 83-85. 45.Gronenborn A.M. et al. , Clore G.M. : (1991). A novel, highly stable fold of the immunoglobulin binding domain of streptococcal protein G. Science 253 : 657-661. 46.Wittekind M. , Gorlach M. , Friedrichs M. , Dreyfuss G. , Mueller L. : (1992). 1H, 13C, and 15N NMR assignments and global folding pattern of the RNA-binding domain of the human hnRNP C proteins. Biochemistry 31 : 6254-6265. 47.Telling G.C. et al. , Prusiner S.B. : (1994). Transmission of Creutzfeldt-Jakob disease from humans to transgenic mice expressing chimeric human-mouse prion protein. Proc. Natl. Acad. Sci. USA 91 : 9936-9940. 48.Bartz J.C. , McKenzie D.I. , Bessen R.A. , Marsh R.F. , Aiken J.M. : (1994). Transmissible mink encephalopathy species barrier effect between ferret and mink: PrP gene and protein analysis. J. Gen. Virol. 75 : 2947-2953. 49.Cervenakova L. et al. , Gajdusek D.C. : (1994). Infectious amyloid precursor gene sequences in primates used for experimental transmission of human spongiform encephalopathy. Proc. Natl. Acad. Sci. USA 91 : 12159-12162. 50.Prusiner S.B. , Hsiao K.K. : (1994). Human prion diseases. Ann. Neurol. 35 : 385-395. 51.Tagliavini F. et al. , Frangione B. : (1993). Synthetic peptides homologous to prion protein residues 106-147 form amyloid-like fibrils in vitro. Proc. Natl. Acad. Sci. USA 90 : 9678-9682. 52.Come J.H. , Fraser P.E. , Lansbury P.T.Jr. : (1993). A kinetic model for amyloid formation in the prion diseases: importance of seeding. Proc. Natl. Acad. Sci. USA 90 : 5959-5963. 53.Nguyen J. , Baldwin M.A. , Cohen F.E. , Prusiner S.B. : (1995). Prion protein peptides induce -helix to ■-sheet conformational transitions. Biochemistry 34 : 4186-4192. 54.Gabriel J.-M. , Oesch B. , Kretzschmar H. , Scott M. , Prusiner S.B. : (1992). Molecular cloning of a candidate chicken prion protein. Proc. Natl. Acad. Sci. USA 89 : 9097-9101. 55.Chou P.Y. , Fasman G.D. : (1978). Empirical predictions of protein conformation. Annu. Rev. Biochem. 47 : 251-276. 56.Garnier J. , Osguthorpe D.J. , Robson B. : (1978). Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J. Mol. Biol. 120 : 97-120. 57.Kneller D.G. , Cohen F.E. , Langridge R. : (1990). Improvement in protein secondary structure prediction by an enhanced neural network. J. Mol. Biol. 214 : 171-182. 58.Rost B. , Sander C. : (1994). Combining evolutionary information and neural networks to predict protein secondary structure. Proteins: Structure Function and Genetics 19 : 55-72. 59.Cohen F.E. , Abarbanel R.M. , Kuntz I.D. , Fletterick R.J. : (1986). Turn prediction in proteins using a pattern-matching approach. Biochemistry 25 : 266-275.