Probing the Structure of the Infectious Amyloid Form of the Prion-forming Domain of HET-s Using High Resolution Hydrogen/Deuterium Exchange Monitored by Mass Spectrometry

The HET-s prion protein of Podospora anserina represents a valuable model system to study the structural basis of prion propagation. In this system, prion infectivity can be generated in vitro from a recombinant protein. We have previously identified the region of the HET-s protein involved in amyloid formation and prion propagation. Herein, we show that a recombinant peptide corresponding to the C-terminal prion-forming domain of HET-s (residues 218–289) displays infectivity. We used high resolution hydrogen/deuterium exchange analyzed by mass spectrometry to gain insight into the structural organization of this infectious amyloid form of the HET-s-(218–289) protein. Deuterium incorporation was analyzed by ion trap mass spectrometry for 76 peptides generated by pepsin proteolysis of HET-s-(218–289). By taking into account sequence overlaps in these peptides, a resolution ranging from 4-amino acids stretches to a single residue could be achieved. This approach allowed us to define highly protected regions alternating with more accessible segments along the HET-s-(218–289) sequence. The HET-s-(218–289) fibrils are thus likely to be organized as a succession of β-sheet segments interrupted by short turns or short loops. The HET-s prion protein of Podospora anserina represents a valuable model system to study the structural basis of prion propagation. In this system, prion infectivity can be generated in vitro from a recombinant protein. We have previously identified the region of the HET-s protein involved in amyloid formation and prion propagation. Herein, we show that a recombinant peptide corresponding to the C-terminal prion-forming domain of HET-s (residues 218–289) displays infectivity. We used high resolution hydrogen/deuterium exchange analyzed by mass spectrometry to gain insight into the structural organization of this infectious amyloid form of the HET-s-(218–289) protein. Deuterium incorporation was analyzed by ion trap mass spectrometry for 76 peptides generated by pepsin proteolysis of HET-s-(218–289). By taking into account sequence overlaps in these peptides, a resolution ranging from 4-amino acids stretches to a single residue could be achieved. This approach allowed us to define highly protected regions alternating with more accessible segments along the HET-s-(218–289) sequence. The HET-s-(218–289) fibrils are thus likely to be organized as a succession of β-sheet segments interrupted by short turns or short loops. Amyloids are fibrillar protein aggregates composed of a “cross-β” structure in which β-strands are oriented perpendicular to the fiber axis. This type of protein aggregate is associated not only with prion diseases but with a variety of protein deposition diseases including Alzheimer disease and Parkinson disease (1.Ross C.A. Poirier M.A. Nat. Med. 2004; 10: S10-S17Crossref PubMed Scopus (2475) Google Scholar). Resolution of the structure of amyloid assemblies is of foremost importance from both a fundamental and a biomedical point of view. As a consequence, much effort has been devoted to the acquisition of structural information on a number of amyloid proteins using a variety of methods including proline scanning mutagenesis (2.Williams A.D. Portelius E. Kheterpal I. Guo J.T. Cook K.D. Xu Y. Wetzel R. J. Mol. Biol. 2004; 335: 833-842Crossref PubMed Scopus (344) Google Scholar, 3.Wood S.J. Wetzel R. Martin J.D. Hurle M.R. Biochemistry. 1995; 34: 724-730Crossref PubMed Scopus (318) Google Scholar), hydrogen exchange (4.Hoshino M. Katou H. Hagihara Y. Hasegawa K. Naiki H. Goto Y. Nat. Struct. Biol. 2002; 9: 332-336Crossref PubMed Scopus (314) Google Scholar, 5.Ippel J.H. Olofsson A. Schleucher J. Lundgren E. Wijmenga S.S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8648-8653Crossref PubMed Scopus (85) Google Scholar, 6.Kheterpal I. Zhou S. Cook K.D. Wetzel R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13597-13601Crossref PubMed Scopus (166) Google Scholar, 7.Kraus M. Bienert M. Krause E. Rapid Commun. Mass Spectrom. 2003; 17: 222-228Crossref PubMed Scopus (28) Google Scholar), limited proteolysis (8.Kheterpal I. Williams A. Murphy C. Bledsoe B. Wetzel R. Biochemistry. 2001; 40: 11757-11767Crossref PubMed Scopus (196) Google Scholar), spin labeling (9.Torok M. Milton S. Kayed R. Wu P. McIntire T. Glabe C.G. Langen R. J. Biol. Chem. 2002; 277: 40810-40815Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar), and solid state NMR (10.Petkova A.T. Ishii Y. Balbach J.J. Antzutkin O.N. Leapman R.D. Delaglio F. Tycko R. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16742-16747Crossref PubMed Scopus (1650) Google Scholar). Various experimental approaches used for structural analysis of amyloid fibrils have been presented in a recent review by Tycko (11.Tycko R. Curr. Opin. Struct. Biol. 2004; 14: 96-103Crossref PubMed Scopus (354) Google Scholar). Among the amyloidogenic proteins, prion proteins possess the unique ability to replicate the amyloid conformation and thus display an infectious character. In mammals, prions are infectious proteinaceous particles that cause fatal neurodegenerative diseases termed spongiform encephalopathies (12.Prusiner S.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13363-13383Crossref PubMed Scopus (5151) Google Scholar). Mammalian prions correspond to an altered form of a cellular protein termed PrP that is converted to a protease-resistant aggregated form in diseased individuals. Proteins capable of propagating an altered conformational state have also been identified in eukaryotic microorganisms (13.Wickner R.B. Science. 1994; 264: 566-569Crossref PubMed Scopus (1086) Google Scholar). The best characterized prion models are the yeast Ure2p and Sup35p proteins and the HET-s protein from the fungus Podospora anserina (14.Wickner R.B. Edskes H.K. Roberts B.T. Baxa U. Pierce M.M. Ross E.D. Brachmann A. Genes Dev. 2004; 18: 470-485Crossref PubMed Scopus (64) Google Scholar). All of these proteins form amyloid fibrils in vitro (15.Glover J.R. Kowal A.S. Schirmer E.C. Patino M.M. Liu J.J. Lindquist S. Cell. 1997; 89: 811-819Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar, 16.Taylor K.L. Cheng N. Williams R.W. Steven A.C. Wickner R.B. Science. 1999; 283: 1339-1343Crossref PubMed Scopus (264) Google Scholar, 17.King C.Y. Tittmann P. Gross H. Gebert R. Aebi M. Wuthrich K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6618-6622Crossref PubMed Scopus (293) Google Scholar, 18.Dos Reis S. Coulary-Salin B. Forge V. Lascu I. Begueret J. Saupe S.J. J. Biol. Chem. 2002; 277: 5703-5706Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). These fungal prion proteins represent valuable models for exploring the mechanism of prion propagation. It is essential to gain insight into the structure of prion proteins in their infectious conformation to elucidate the details of prion replication and also to determine whether specific structural features are associated with the infectious character. The HET-s prion protein of P. anserina is involved in a genetically programmed cell death reaction termed heterokaryon incompatibility. This cell death reaction is triggered when the prion form of HET-s interacts with a natural variant of HET-s, designated HET-S, which differs from HET-s by 13 residues and is devoid of prion behavior (19.Coustou V. Deleu C. Saupe S. Begueret J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9773-9778Crossref PubMed Scopus (407) Google Scholar). HET-s aggregates specifically in vivo upon transition to the prion state (20.Coustou-Linares V. Maddelein M.L. Begueret J. Saupe S.J. Mol. Microbiol. 2001; 42: 1325-1335Crossref PubMed Scopus (49) Google Scholar). Recombinant HET-s forms typical amyloid fibrils in vitro (18.Dos Reis S. Coulary-Salin B. Forge V. Lascu I. Begueret J. Saupe S.J. J. Biol. Chem. 2002; 277: 5703-5706Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). We were able to show that introduction of amyloid aggregates of recombinant HET-s into P. anserina cells induces the [Het-s] prion with a very high efficiency (21.Maddelein M.L. Dos Reis S. Duvezin-Caubet S. Coulary-Salin B. Saupe S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7402-7407Crossref PubMed Scopus (236) Google Scholar). Hence, the amyloids generated in vitro represent infectious material that can propagate the [Het-s] prion. The HET-s protein is 289 amino acids in length and is composed of two distinct domains: an N-terminal globular domain spanning approximately residues 1 to 230 and a flexible domain spanning approximately residues 230 to 289 (22.Balguerie A. Dos Reis S. Ritter C. Chaignepain S. Coulary-Salin B. Forge V. Bathany K. Lascu I. Schmitter J.M. Riek R. Saupe S.J. EMBO J. 2003; 22: 2071-2081Crossref PubMed Scopus (171) Google Scholar). In the amyloid form of HET-s, the C-terminal domain of HET-s forms the protease-resistant amyloid core of the fibril, whereas the N-terminal domain remains accessible to proteolysis. The C-terminal domain of HET-s (residues 218–289) is sufficient for [Het-s] propagation in vivo and amyloid formation in vitro (22.Balguerie A. Dos Reis S. Ritter C. Chaignepain S. Coulary-Salin B. Forge V. Bathany K. Lascu I. Schmitter J.M. Riek R. Saupe S.J. EMBO J. 2003; 22: 2071-2081Crossref PubMed Scopus (171) Google Scholar). Hydrogen/deuterium exchange combined with mass spectrometry (HXMS) 1The abbreviations used are: HXMS, hydrogen/deuterium exchange combined with mass spectrometry; H/D, hydrogen/deuterium; PrP, prion protein; H2O MQ, Ultrapure milli-Q H2O; MS, mass spectroscopy; LC-MS/MS, liquid chromatography-tandem mass spectroscopy. has become a powerful tool for the study of protein structures and dynamics (3.Wood S.J. Wetzel R. Martin J.D. Hurle M.R. Biochemistry. 1995; 34: 724-730Crossref PubMed Scopus (318) Google Scholar, 23.Smith D.L. Deng Y. Zhang Z. J. Mass Spectrom. 1997; 32: 135-146Crossref PubMed Scopus (385) Google Scholar, 24.Wagner D.S. Melton L.G. Yan Y. Erickson B.W. Anderegg R.J. Protein Sci. 1994; 3: 1305-1314Crossref PubMed Scopus (57) Google Scholar, 25.Engen J.R. Smith D.L. Anal. Chem. 2001; 73: 256A-265ACrossref PubMed Google Scholar, 26.Maier C.S. Schimerlik M.I. Deinzer M.L. Biochemistry. 1999; 38: 1136-1143Crossref PubMed Scopus Google Scholar, P. J. Mol. Biol. 2000; PubMed Scopus Google Scholar). This the ability of mass spectrometry to determine the incorporation of in proteins a reaction of exchange with exchange for on hydrogen and to the This exchange also on the and the of the exchange Protein dynamics can be by hydrogen exchange and on the and of the protein Y. Protein Sci. 1997; PubMed Scopus Google Scholar, S. Curr. Opin. 1998; 9: PubMed Scopus Google Scholar). a of in specific the protein can be to proteolysis the exchange reaction Z. Smith D.L. Protein Sci. PubMed Scopus Google Scholar). details are presented in a review by and Smith J.R. Smith D.L. Anal. Chem. 2001; 73: 256A-265ACrossref PubMed Google Scholar). Hydrogen/deuterium exchange combined with mass spectrometry or NMR has been used to the structural of amyloid peptides or proteins (4.Hoshino M. Katou H. Hagihara Y. Hasegawa K. Naiki H. Goto Y. Nat. Struct. Biol. 2002; 9: 332-336Crossref PubMed Scopus (314) Google Scholar, 5.Ippel J.H. Olofsson A. Schleucher J. Lundgren E. Wijmenga S.S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 8648-8653Crossref PubMed Scopus (85) Google Scholar, K. T. Cheng H. K. H. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar, K. Katou H. M. Hasegawa K. Naiki H. Goto Y. J. Mol. Biol. 2004; PubMed Scopus Google Scholar, I. T. Wetzel R. Biochemistry. 2003; 42: PubMed Scopus Google Scholar, A. Dos Reis S. M. Saupe S.J. Schmitter J.M. Biochemistry. 2003; 42: PubMed Scopus Google Scholar). It has been that in core regions of amyloid aggregates are highly to hydrogen exchange (4.Hoshino M. Katou H. Hagihara Y. Hasegawa K. Naiki H. Goto Y. Nat. Struct. Biol. 2002; 9: 332-336Crossref PubMed Scopus (314) Google Scholar, 6.Kheterpal I. Zhou S. Cook K.D. Wetzel R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13597-13601Crossref PubMed Scopus (166) Google Scholar, 7.Kraus M. Bienert M. Krause E. Rapid Commun. Mass Spectrom. 2003; 17: 222-228Crossref PubMed Scopus (28) Google Scholar). exchange can thus be used to the number of involved in a highly Kheterpal I. Wetzel R. Cook K.D. Protein Sci. 2003; PubMed Scopus Google have exchange for that of are highly protected from exchange can also be used to highly peptide segments from regions that are not involved in structure protected regions are as β-sheet core whereas regions of exchange are as or analysis by hydrogen spectrometry has that the prion-forming domain of HET-s protein is highly protected from hydrogen exchange in the amyloid form A. Dos Reis S. M. Saupe S.J. Schmitter J.M. Biochemistry. 2003; 42: PubMed Scopus Google Scholar). we have by of a that the amyloid form of the recombinant HET-s-(218–289) peptide can the [Het-s] prion when in We used high resolution to gain structural information on the infectious amyloid form of this HET-s-(218–289) and HET-s-(218–289) peptide was and from as previously (22.Balguerie A. Dos Reis S. Ritter C. Chaignepain S. Coulary-Salin B. Forge V. Bathany K. Lascu I. Schmitter J.M. Riek R. Saupe S.J. EMBO J. 2003; 22: 2071-2081Crossref PubMed Scopus (171) Google Scholar). the peptide was to on a using as an Protein was to the amyloid of was to by the of formation was by and by as previously (22.Balguerie A. Dos Reis S. Ritter C. Chaignepain S. Coulary-Salin B. Forge V. Bathany K. Lascu I. Schmitter J.M. Riek R. Saupe S.J. EMBO J. 2003; 22: 2071-2081Crossref PubMed Scopus (171) Google Scholar). amyloid aggregates were by and aggregates were in infectivity was as previously (21.Maddelein M.L. Dos Reis S. Duvezin-Caubet S. Coulary-Salin B. Saupe S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7402-7407Crossref PubMed Scopus (236) Google Scholar). of the the HET-s protein in form were for on solid were of or HET-s protein was in a of of of the were with of particles in a system using were for and two were from with a In this that the a with the whereas that the [Het-s] prion an designated and from the cell death of the two a reaction with the the was as for the [Het-s] prion of Deuterium in of aggregated HET-s-(218–289) in Ultrapure milli-Q H2O was for The was with 1 of H2O of the aggregated was in an from to of the protein was to a The of was into a to ion trap mass Mass of HET-s-(218–289) HET-s-(218–289) H2O was for The was with 1 of H2O of the aggregated was in of the was in with pepsin and for was by a short and a of the the HET-s was to HET-s-(218–289) peptides were analyzed with an ion trap mass to a system the was used to peptide from their on HET-s-(218–289) HET-s-(218–289) H2O was for The was with 1 of H2O of the aggregated was in and for and of the was in with pepsin and was by a short and a of the the HET-s was into by from a The were with of a and of was into a to mass The from to was for Mass in the for exchange correspond to of the of the of a ion and a pepsin of HET-s-(218–289) was in and for to exchange of for the exchange was by the of the peptide was on a The peptide from the was a with was into a mass The was for peptide by the number of hydrogen and the corresponding experimental a pepsin was in the MQ, The peptide was on a and with a with The incorporation of for was in the as the exchange on the ion trap the were in the by of The was to The was to of the protein used for exchange on amyloid was a with a was allowed to in a were with of in with and with a were and the number of in a peptide was from the of using the in 1 Y. Smith D.L. J. Mass Spectrom. 1994; PubMed Scopus Google Scholar). is the mass of the peptide point is the mass point peptides, is the mass for a peptide and is the number of peptide in the The of incorporation is in for peptides of the peptide mass Amyloids of HET-s-(218–289) amyloid core of HET-s amyloids to the region spanning residue (22.Balguerie A. Dos Reis S. Ritter C. Chaignepain S. Coulary-Salin B. Forge V. Bathany K. Lascu I. Schmitter J.M. Riek R. Saupe S.J. EMBO J. 2003; 22: 2071-2081Crossref PubMed Scopus (171) Google Scholar, A. Dos Reis S. Coulary-Salin B. Chaignepain S. M. Schmitter J.M. Saupe S.J. J. Sci. 2004; PubMed Scopus Google Scholar). We have previously that HET-s fibrils to infectivity (21.Maddelein M.L. Dos Reis S. Duvezin-Caubet S. Coulary-Salin B. Saupe S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7402-7407Crossref PubMed Scopus (236) Google Scholar). these that HET-s-(218–289) amyloids generated in vitro be It that infectivity of the HET-s amyloids could be to an of protein or that the HET-s-(218–289) domain a structure in HET-s amyloid as in amyloids from the HET-s-(218–289) that amyloid aggregates of HET-s-(218–289) display an infectious we aggregates into cells using the previously used The HET-s-(218–289) peptide was for were by As previously these HET-s-(218–289) typical amyloid By HET-s-(218–289) amyloids as fibrils of in HET-s-(218–289) amyloids were on the of a of a on solid were with particles as previously (21.Maddelein M.L. Dos Reis S. Duvezin-Caubet S. Coulary-Salin B. Saupe S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7402-7407Crossref PubMed Scopus (236) Google to the recombinant peptide into the fungal the were for the of the [Het-s] prion in the that were in the of HET-s-(218–289) the [Het-s] prion. In the with HET-s of the We from this as previously for the HET-s amyloid aggregates of the recombinant HET-s-(218–289) peptide are introduction of the HET-s-(218–289) peptide in Podospora induces the [Het-s] prion. have been with HET-s or HET-s-(218–289) with and for the of the [Het-s] prion by the with of a in the that the has the [Het-s] of Deuterium in HET-s-(218–289) exchange was allowed to on HET-s-(218–289) amyloid aggregates for from to The was to the of in the which to of the of the protein. a in of the hydrogen of HET-s-(218–289) are protected in the amyloid was for the incorporation of of exchange we to study the of aggregated HET-s protein this This that a of the residues are highly protected from thus the with the HET-s protein A. Dos Reis S. M. Saupe S.J. Schmitter J.M. Biochemistry. 2003; 42: PubMed Scopus Google Scholar). These with by Kheterpal I. Wetzel R. Cook K.D. Protein Sci. 2003; PubMed Scopus Google for in their of in of were highly protected from Mass of proteolysis of HET-s-(218–289) by pepsin a number of peptides could be to their sequence. These peptides the sequence of the HET-s-(218–289) protein to a by the were in the to the pepsin was by with a on a In this peptides of the peptides by could be in the 76 peptides a of sufficient for in to determine the incorporation in HET-s-(218–289) on the of the HET-s-(218–289) of HET-s-(218–289) were as and amyloid formation was by were for in the of have been to determine the incorporation of in aggregated HET-s-(218–289) this and the incorporation of was for In the of the pepsin the of incorporation for peptide In the the of incorporation for peptide exchange The incorporation along the HET-s-(218–289) sequence was to be very peptides exchange were as as or as high as incorporation was in peptides corresponding to the N-terminal region of HET-s-(218–289) and in C-terminal of to Resolution of Deuterium the resolution of incorporation for the study of we have the of of 76 peptides by taking into account their as by Zhang and Smith Z. Smith D.L. Protein Sci. PubMed Scopus Google Scholar). By of peptides, regions were with a resolution ranging from 1 to amino acids we could determine the incorporation of by incorporation in peptides and for and and the exchange HET-s-(218–289) protein was for in pepsin proteolysis. the was analyzed using and ion trap mass mass for mass for These peptides are as the number of is for both peptides, of the incorporation of the Mass for the exchange correspond to of the of the of the mass for This approach designated the regions and as the accessible to the with exchange to or The regions and the C-terminal are the with incorporation In regions the exchange to or as for in or with residues and were also identified have to a of the resolution of for the aggregated HET-s prion protein. The amyloid infectious form of the HET-s-(218–289) peptide is organized as a succession of segments of length with alternating high and exchange of that prion proteins propagate as amyloid aggregates in both and N. Mol. Cell. 2004; 14: Full Text Full Text PDF PubMed Scopus Google Scholar). a point that remains is a prion protein aggregate from amyloid The structural of the infectious form of prion proteins is essential to that and to the mechanism infectious propagation of an amyloid The fungal [Het-s] prion is for the prion form of the HET-s protein can be in vitro from a recombinant protein (21.Maddelein M.L. Dos Reis S. Duvezin-Caubet S. Coulary-Salin B. Saupe S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7402-7407Crossref PubMed Scopus (236) Google Scholar). we the of the C-terminal of HET-s, designated in in vivo as the prion-forming as a model system for the structural of a prion. a to recombinant protein into we have that a recombinant peptide corresponding to the of HET-s displays infectivity in amyloid These that HET-s infectivity is with and that the core of HET-s residues (21.Maddelein M.L. Dos Reis S. Duvezin-Caubet S. Coulary-Salin B. Saupe S.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7402-7407Crossref PubMed Scopus (236) Google Scholar, A. Dos Reis S. Ritter C. Chaignepain S. Coulary-Salin B. Forge V. Bathany K. Lascu I. Schmitter J.M. Riek R. Saupe S.J. EMBO J. 2003; 22: 2071-2081Crossref PubMed Scopus (171) Google Scholar). the that infectivity of a be to an of HET-s protein. In that the region can an infectious amyloid in vitro whether or not is to the HET-s globular This that the globular domain not the acquisition of the infectious amyloid in These the HET-s-(218–289) peptide a very model for the structural of a prion protein. In vitro propagation of was with a protein residues of to the protein C.Y. R. 2004; PubMed Scopus Google Scholar). It is thus likely that infectious be in the to the HET-s-(218–289) peptide to the peptide for which prion infectivity has been gain structural information on the organization of the HET-s-(218–289) in amyloid form we this by of Deuterium incorporation was for 76 peptides spanning the sequence of residues of these peptides were used to incorporation for short We thus exchange for of residues resolution of residues was exchange the amino resolution only for a limited number of in short segments the exchange is an and incorporation the residue be very short peptide regions as or are not by the identified peptides, and we exchange information for The that were used were to resolution The of exchange high resolution using also to only in the exchange are these approach a to structural information on the organization of the HET-s-(218–289) This approach is to a of amyloid or prion proteins and accessible to as become more we have used a exchange the exchange by the study is This that the HET-s-(218–289) structure is very and this domain displays a to proteolysis. The is that the incorporation is not along the peptide sequence. are in segments or in peptides and can be as as In the of structural be that distinct structures an amyloid of HET-s-(218–289). the that incorporation are along the HET-s-(218–289) sequence that amyloid display only limited structural or that structural variant of amyloid aggregates region of exchange are as structural whereas accessible regions exchange have been to correspond to regions that are not involved in structure (11.Tycko R. Curr. Opin. Struct. Biol. 2004; 14: 96-103Crossref PubMed Scopus (354) Google Scholar). regions with very exchange have been for segments and These regions likely correspond to In more accessible regions for could correspond to turns or short loops. The HET-s structure could thus be organized as a succession of segments interrupted by turns or loops. protected segments and but of the resolution the of these be In not the of structural but also be the of the of the the M. Bienert M. Krause E. Rapid Commun. Mass Spectrom. 2003; 17: 222-228Crossref PubMed Scopus (28) Google Scholar, the identified protected segments represent for It is to to a structural model from the but can be as exchange along the can be that the HET-s-(218–289) amyloid is organized as an a of in an a model is also with the of HET-s-(218–289) fibrils by regions characterized by a very exchange are short amino as segments exchange This that the segments are interrupted by turns or models have been for prion proteins or protein In the of PrP, combined with have to the that PrP assemblies a structure H. V. S. A. S.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar, C. H. S.B. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar). In the structure of PrP, segments are interrupted by turns or loops. model has also been for the yeast prion domain on the basis of fiber A. Hasegawa K. H. H. K. M. Commun. 2004; PubMed Scopus Google Scholar, J.T. J. A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: PubMed Scopus Google Scholar). These models that forms a composed of an be with a model the model with an β-sheet structure exchange are not along the sequence. the model is that β-sheet regions are interrupted by turns and the exchange in or model on a organization has been for the yeast Ure2p prion domain Baxa U. Wickner R.B. Steven A.C. Proc. Natl. Acad. Sci. U. S. A. 2004; PubMed Scopus Google Scholar). This model also be for the prion domain and amyloid in HET-s the protected regions are not and are interrupted by more the model to HET-s from the organization for the and the turns be in the Ure2p In the approach allowed us to gain into the structural features of the HET-s-(218–289) peptide in infectious amyloid This structural information and thus to the of the structural basis of prion infectivity in this model The approach that we also be in of conformational as in yeast prions C.Y. R. 2004; PubMed Scopus Google Scholar, M. P. N. R. 2004; PubMed Scopus Google Scholar).