Two Amyloid States of the Prion Protein Display Significantly Different Folding Patterns

It has been well established that a single amino acid sequence can give rise to several conformationally distinct amyloid states. The extent to which amyloid structures formed within the same sequence are different, however, remains unclear. To address this question, we studied two amyloid states (referred to as R- and S-fibrils) produced in vitro from highly purified full-length recombinant prion protein. Several biophysical techniques including X-ray diffraction, CD, Fourier transform infrared spectroscopy (FTIR), hydrogen-deuterium exchange, proteinase K digestion, and binding of a conformation-sensitive fluorescence dye revealed that R- and S-fibrils have substantially different secondary, tertiary, and quaternary structures. While both states displayed a 4. 8-Å meridional X-ray diffraction typical for amyloid cross-β-spines, they showed markedly different equatorial profiles, suggesting different folding pattern of β-strands. The experiments on hydrogen-deuterium exchange monitored by FTIR revealed that only small fractions of amide protons were protected in R- or S-fibrils, an argument for the dynamic nature of their cross-β-structure. Despite this fact, both amyloid states were found to be very stable conformationally as judged from temperature-induced denaturation monitored by FTIR and the conformation-sensitive dye. Upon heating to 80 °C, only local unfolding was revealed, while individual state-specific cross-β features were preserved. The current studies demonstrated that the two amyloid states formed by the same amino acid sequence exhibited significantly different folding patterns that presumably reflect two different architectures of cross-β-structure. Both S- and R-fibrils, however, shared high conformational stability, arguing that the energy landscape for protein folding and aggregation can contain several deep free-energy minima.     

Characterization and enzymatic degradation of Sup35NM, a yeast prion-like protein

Transmissible spongiform encephalopathies (TSEs) are believed to be caused by an unconventional infectious agent, the prion protein. The pathogenic and infectious form of prion protein, PrPSc, is able to aggregate and form amyloid fibrils, very stable and resistant to most disinfecting processes and common proteases. Under specific conditions, PrPSc in bovine spongiform encephalopathy (BSE) brain tissue was found degradable by a bacterial keratinase and some other proteases. Since this disease-causing prion is infectious and dangerous to work with, a model or surrogate protein that is safe is needed for the in vitro degradation study. Here a nonpathogenic yeast prion-like protein, Sup35NM, cloned and overexpressed in E. coli, was purified and characterized for this purpose. Aggregation and deaggregation of Sup35NM were examined by electron microscopy, gel electrophoresis, Congo red binding, fluorescence, and Western blotting. The degradation of Sup35NM aggregates by keratinase and proteinase K under various conditions was studied and compared. These results will be of value in understanding the mechanism and optimization of the degradation process.     

DNA-induced partial unfolding of prion protein leads to its polymerisation to amyloid

The full-length mouse recombinant prion protein (23-231 amino acid residues) contains all of its structural elements viz. three alpha-helices and a short two-stranded antiparallel beta-sheet in its C-terminal fragment comprising 121-231 amino acid residues. The incubated mixture of this prion protein fragment and nucleic acid results in the formation of amyloid fibres evidenced from electron microscopy, birefringence and fluorescence of the fibre bound Congo Red and Thioflavin T dyes, respectively. The secondary structure of the amyloid formed in nucleic acid solution is similar to the in vivo isolated prion protein 27-30 amyloid but unlike in it, a hydrophobic milieu is absent in the 121-231 amyloid. Thermal denaturation study demonstrates a partial unfolding of the protein fragment in nucleic acid solution. We propose that nucleic acid catalyses unfolding of prion protein helix 1 followed by a nucleation-dependent polymerisation of the protein to amyloid.     

Structural Insights into Alternate Aggregated Prion Protein Forms

The conversion of the cellular form of the prion protein (PrP^C) to an abnormal, alternatively folded isoform (PrP^Sc) is the central event in prion diseases or transmissible spongiform encephalopathies. Recent studies have demonstrated de novo generation of murine prions from recombinant prion protein (recPrP) after inoculation into transgenic and wild-type mice. These so-called synthetic prions lead to novel prion diseases with unique neuropathological and biochemical features. Moreover, the use of recPrP in an amyloid seeding assay can specifically detect and amplify various strains of prions. We employed this assay in our experiments and analyzed in detail the morphology of aggregate structures produced under defined chemical constraints. Our results suggest that changes in the concentration of guanidine hydrochloride can lead to different kinetic traces in a typical thioflavin T(ThT) assay. Morphological and structural analysis of these aggregates by atomic force microscopy indicates a variation in the structure of the PrP molecular assemblies.In particular, ThT positive PrP aggregates produced from rec mouse PrP residues 89 to 230 lead to mostly oligomeric structures at low concentrations of guanidine hydrochloride, while more amyloidal structures were observed at higher concentrations of the denaturant. These findings highlight the presence of numerous and complex pathways in deciphering prion constraints for infectivity and toxicity.     

Sequential generation of two structurally distinct ovine prion protein soluble oligomers displaying different biochemical reactivities

In pathologies due to protein misassembly, low oligomeric states of the misfolded proteins rather than large aggregates play an important biological role. In prion diseases the lethal evolution is associated with formation of PrP(Sc), a misfolded and amyloid form of the normal cellular prion protein PrP. Although several molecular mechanisms were proposed to account for the propagation of the infectious agent, the events responsible for cell death are still unclear. The correlation between PrP(C) expression level and the rate of disease evolution on one side, and the fact that PrP(Sc) deposition in brain did not strictly correlate with the apparition of clinical symptoms on the other side, suggested a potential role for diffusible oligomers in neuronal death. To get better insight into the molecular mechanisms of PrP(C) oligomerization, we studied the heat-induced oligomerization pathway of the full-length recombinant ovine PrP at acidic pH. This led to the irreversible formation of two well-identified soluble oligomers that could be recovered by size-exclusion chromatography. Both oligomers displayed higher beta-sheet content when compared to the monomer. A sequential two-step multimolecular process accounted for the rate of their formation and their ratio partition, both depending on the initial protein concentration. Small-angle X-ray scattering allowed the determination of the molecular masses for each oligomer, 12mer and 36mer, as well as their distinct oblate shapes. The two species differed in accessibility of polypeptide chain epitopes and of pepsin-sensitive bonds, in a way suggesting distinct conformations for their monomeric unit. The conversion pathway leading to these novel oligomers, displaying contrasted biochemical reactivities, might be a clue to unravel their biological roles.     

Inter-oligomer interactions of the human prion protein are modulated by the polymorphism at codon 129

The common polymorphism at codon 129 in the human prion protein (PrP) has been shown in many studies to influence not only the pathology of prion disease but also the misfolding propensity of PrP. Here we used NMR, CD and atomic force microscopy in solution to investigate differences in β-oligomer (β^O) formation and inter-oligomer interaction depending on the polymorphism at codon 129. NMR investigations assigned the observable amide resonances to the β^O N-terminal segments, showing that it is the core region of PrP (residues 127-228) that is involved in β^O formation. Atomic force microscopy revealed distinctive 1.8 × 15 × 15-nm disk-like structures that form stacks through inter-oligomer interactions. The propensity to form stacks and the number of oligomers involved depended on the polymorphism at codon 129, with a significantly lower degree of stacking for β^O with valine at position 129. This result provides evidence for conformational differences between the β^O allelic forms, showing that the core region of the protein including position 129 is actively involved in inter-oligomer interactions, consistent with NMR observations.     

The polybasic N-terminal region of the prion protein controls the physical properties of both the cellular and fibrillar forms of PrP

Individual variations in structure and morphology of amyloid fibrils produced from a single polypeptide are likely to underlie the molecular origin of prion strains and control the efficiency of the species barrier in the transmission of prions. Previously, we observed that the shape of amyloid fibrils produced from full-length prion protein (PrP 23-231) varied substantially for different batches of purified recombinant PrP. Variations in fibril morphology were also observed for different fractions that corresponded to the highly pure PrP peak collected at the last step of purification. A series of biochemical experiments revealed that the variation in fibril morphology was attributable to the presence of miniscule amounts of N-terminally truncated PrPs, where a PrP encompassing residue 31-231 was the most abundant of the truncated polypeptides. Subsequent experiments showed that the presence of small amounts of recombinant PrP 31-231 (0.1-1%) in mixtures with full-length PrP 23-231 had a dramatic impact on fibril morphology and conformation. Furthermore, the deletion of the short polybasic N-terminal region 23-30 was found to reduce the folding efficiency to the native α-helical forms and the conformational stability of α-PrP. These findings are very surprising considering that residues 23-30 are very distant from the C-terminal globular folded domain in α-PrP and from the prion folding domain in the fibrillar form. However, our studies suggest that the N-terminal polybasic region 23-30 is essential for effective folding of PrP to its native cellular conformation. This work also suggests that this region could regulate diversity of prion strains or subtypes despite its remote location from the prion folding domain.     

FTIR-microspectroscopy of prion-infected nervous tissue

The family of transmissible spongiform encephalopathies (TSE), also termed prion diseases, is a group of fatal, neurodegenerative diseases characterized by the accumulation of a misfolded protein, the disease-associated prion protein PrP^Sc. This glycoprotein differs in secondary structure from its normal, cellular isoform PrP^C, which is physiologically expressed mostly by neurons. Scrapie is a prion disease first described in the 18th century in sheep and goats, and has been established as a model in rodents to study the pathogenesis and pathology of prion diseases. Assuming a multitude of molecular parameters change in the tissue in the course of the disease, FTIR microspectroscopy has been proposed as a valuable new method to study and identify prion-affected tissues due to its ability to detect a variety of changes in molecular structure and composition simultaneously. This paper reviews and discusses results from previous FTIR microspectroscopic studies on nervous tissue of scrapie-infected hamsters in the context of histological and molecular alterations known from conventional pathogenesis studies. In particular, data from studies reporting on disease-specific changes of protein structure characteristics, and also results of a recent study on hamster dorsal root ganglia (DRG) are discussed. These data include an illustration on how the application of a brilliant IR synchrotron light source enables the in situ investigation of localized changes in protein structure and composition in nervous cells or tissue due to PrP^Sc deposition, and a demonstration on how the IR spectral information can be correlated with results of complementary studies using immunohistochemistry and x-ray fluorescence techniques. Using IR microspectroscopy, some neurons exhibited a high accumulation of disease-associated prion protein evidenced by an increased amount of β-sheet at narrow regions in or around the infected nervous cells. However, not all neurons from terminally diseased hamsters showed PrP^Sc deposition. Generally, the average spectral differences between all control and diseased DRG spectra are small but consistent as demonstrated by independent experiments. Along with studies on the purified misfolded prion protein, these data suggest that synchrotron FTIR microspectroscopy is capable of detecting the misfolded prion protein in situ without the necessity of immunostaining or purification procedures.     

Potential approaches for heterologous prion protein treatment of prion diseases

Prion diseases, or transmissible spongiform encephalopathies (TSEs) are progressive, fatal neurodegenerative diseases with no effective treatment. The pathology of these diseases involves the conversion of a protease sensitive form of the cellular prion protein (PrP^C) into a protease resistant infectious form (PrP^res). The efficiency of this conversion is predicated upon a number of factors, most notably a strong homology between cellular PrP^C and PrP^res. In our recently published study, we infected mice with the RML-Chandler strain of scrapie and treated them with heterologous hamster prion proteins. This treatment was seen to reduce clinical signs of prion disease, to delay the onset of clinical symptoms and to prolong survival. In this current article we discuss potential mechanisms of action of treatment with heterologous prion proteins. We also discuss potential extensions of these studies using a heterologous rabbit PrP-based treatment strategy or a peptide based strategy, and improvement of treatment delivery including a lentiviral-based system.     

On the mechanism of alpha-helix to beta-sheet transition in the recombinant prion protein

It is believed that the critical event in the pathogenesis of transmissible spongiform encephalopathies is the conversion of the prion protein from an alpha-helical form, PrP(C), to a beta-sheet-rich conformer, PrP(Sc). Recently, we have shown that incubation of the recombinant prion protein under mildly acidic conditions (pH 5 or below) in the presence of low concentrations of guanidine hydrochloride results in a transition to PrP(Sc)-like beta-sheet-rich oligomers that show fibrillar morphology and an increased resistance to proteinase K digestion [Swietnicki, W., Morillas, M, Chen, S., Gambetti, P., and Surewicz, W. K. (2000) Biochemistry 39, 424-431]. To gain insight into the mechanism of this transition, in the present study we have characterized the biophysical properties of the recombinant human prion protein (huPrP) at acidic pH in the presence of urea and salt. Urea alone induces unfolding of the protein but does not result in protein self-association or a conversion to beta-sheet structure. However, a time-dependent transition to beta-sheet structure occurs upon addition of both urea and NaCl to huPrP, even at a sodium chloride concentration as low as 50 mM. This transition occurs concomitantly with oligomerization of the protein. At a given protein and sodium chloride concentration, the rate of monomeric alpha-helix to oligomeric beta-sheet transition is strongly dependent on the concentration of urea. Low and medium concentrations of the denaturant accelerate the reaction, whereas strongly unfolding conditions are not conducive to the conversion of huPrP into an oligomeric beta-sheet-rich structure. The present data strongly suggest that partially unfolded intermediates may be involved in the transition of the monomeric recombinant prion protein into the oligomeric scrapie-like form.     

Nucleic acid induced unfolding of recombinant prion protein globular fragment is pH dependent

Nucleic acid can catalyze the conversion of α‐helical cellular prion protein to β‐sheet rich Proteinase K resistant prion protein oligomers and amyloid polymers in vitro and in solution. Because unfolding of a protein molecule from its ordered α‐helical structure is considered to be a necessary step for the structural conversion to its β‐sheet rich isoform, we have studied the unfolding of the α‐helical globular 121–231 fragment of mouse recombinant prion protein in the presence of different nucleic acids at neutral and acid pH. Nucleic acids, either single or double stranded, do not have any significant effect on the secondary structure of the protein fragment at neutral pH; however the protein secondary structure is modified by the nucleic acids at pH 5. Nucleic acids do not show any significant effect on the temperature induced unfolding of the globular prion protein domain at neutral pH which, however, undergoes a gross conformational change at pH 5 as evidenced from the lowering of the midpoint of thermal denaturation temperatures, Tm, of the protein. The extent of Tm decrease shows a dependence on the nature of nucleic acid. The interaction of nucleic acid with the nonpolar groups exposed from the protein interior at pH 5 probably contributes substantially to the unfolding process of the protein.     

Thermodynamic characterization for the denatured state of bovine prion protein and the BSE Associated variant E211K

ABSTRACT

Propagation of transmissible spongiform encephalopathies involves the conversion of cellular prion protein, PrP^C, into a misfolded oligomeric form, PrP^Sc. The most common hereditary prion disease is a genetic form of Creutzfeldt-Jakob disease in humans, in which a mutation in the prion gene results in a glutamic acid to lysine substitution at position 200 (E200K) in PrP. In cattle, the analogous amino acid substitution is found at residue 211 (E211K) and has been associated with a case of bovine spongiform encephalopathy. Here, we have compared the secondary structure of E211K to that of wild type using circular dichroism and completed a thermodynamic analysis of the folding of recombinant wild type and E211K variants of the bovine prion protein. The secondary structure of the E211K variant was essentially indistinguishable from that of wild type. The thermodynamic stability of E211K substitution showed a slight destabilization relative to the wild type consistent with results reported for recombinant human prion protein and its mutant E200K. In addition, the E211K variant exhibits a similarly compact denatured state to that of wild type based upon similar m-value and change in heat capacity of unfolding for the proteins. Together these results indicate that residual structure in the denatured state of bPrP is present in both the wild type protein and BSE associated variant E211K. Given this observation, as well as folding similarities reported for other disease associated variants of PrP it is worth consideration that functional aspects of PrP conformation may play a role in the misfolding process.     

Elongated oligomers assemble into mammalian PrP amyloid fibrils

In prion diseases, the mammalian prion protein PrP is converted from a monomeric, mainly alpha-helical state into beta-rich amyloid fibrils. To examine the structure of the misfolded state, amyloid fibrils were grown from a beta form of recombinant mouse PrP (residues 91-231). The beta-PrP precursors assembled slowly into amyloid fibrils with an overall helical twist. The fibrils exhibit immunological reactivity similar to that of ex vivo PrP Sc. Using electron microscopy and image processing, we obtained three-dimensional density maps of two forms of PrP fibrils with slightly different twists. They reveal two intertwined protofilaments with a subunit repeat of approximately 60 A. The repeating unit along each protofilament can be accounted for by elongated oligomers of PrP, suggesting a hierarchical assembly mechanism for the fibrils. The structure reveals flexible crossbridges between the two protofilaments, and subunit contacts along the protofilaments that are likely to reflect specific features of the PrP sequence, in addition to the generic, cross-beta amyloid fold.     

Amyloidogenic unfolding intermediates differentiate sheep prion protein variants

Sheep is a unique example among mammalian species to present a strong correlation between genotype and prion disease susceptibility phenotype. Indeed a well-defined set of PrP polymorphisms at positions 136, 154 and 171 (sheep numbering) govern scrapie susceptibility, ranging from very high susceptibility for V136-R154-Q171 variant (VRQ) to resistance for A136-R154-R171 variant (ARR).To get better insight into the molecular mechanisms of scrapie susceptibility/resistance, the unfolding pathways of the different full-length recombinant sheep prion protein variants were analysed by differential scanning calorimetry in a wide range of pH. In the pH range 4.5-6.0, thermal unfolding occurs through a reversible one-step process while at pH <4.5 and >6.0 unfolding intermediates are formed, which are stable in the temperature range 65-80 degrees C. While these general behaviours are shared by all variants, VRQ and ARQ (susceptibility variants) show higher thermal stability than AHQ and ARR (resistance variants) and the formation of their unfolding intermediates requires higher activation energy than in the case of AHQ and ARR. Furthermore, secondary structures of the unfolding intermediates differentiate variants: ARR unfolding intermediate exhibits random coil structure, contrasting with the beta-sheet structure of VRQ and ARQ unfolding intermediates. The rate of the unfolding intermediate formation allows us to classify genetic variants along increasing scrapie susceptibility at pH 4.0, VRQ and ARQ rates being the highest. Rather poor correlation is observed at pH 7.2. Upon cooling, these intermediates refold into stable species, which are rich in beta-type secondary structures and, as revealed by thioflavin T fluorescence and electron microscopy, share amyloid characteristics. These results highlight the prion protein plasticity genetically modulated in sheep, and might provide a molecular basis for sheep predisposition to scrapie taking into account both thermodynamic stability and transconformation rate of prion protein.     

Amino terminal interaction in the prion protein identified using fusion to green fluorescent protein

In contrast to the well-characterized carboxyl domain, the amino terminal half of the mature cellular prion protein has no defined structure. Here, following fusion of mouse prion protein fragments to green fluorescence protein as a reporter of protein stability, we report extreme variability in fluorescence level that is dependent on the prion fragment expressed. In particular, exposure of the extreme amino terminus in the context of a truncated prion protein molecule led to rapid degradation, whereas the loss of only six amino terminal residues rescued high level fluorescence. Study of the precise endpoints and residue identity associated with high fluorescence suggested a domain within the amino terminal half of the molecule defined by a long-range intramolecular interaction between 23KKRPKP28 and 143DWED146 and dependent upon the anti-parallel beta-sheet ending at residue 169 and normally associated with the structurally defined carboxyl terminal domain. This previously unreported interaction may be significant for understanding prion bioactivity and for structural studies aimed at the complete prion structure.     

Structure, dynamics, and stability of assemblies of the human prion fragment SNQNNF

Misfolding of the prion protein (PrP) is associated with the development of Transmissible Spongiform Encephalopathies. The recent crystal structure of ‘steric zipper’ aggregates of the peptide SNQNNF (human PrP fragment 170-175) has highlighted its potential involvement in the misfolding process. A detailed molecular dynamics investigation on SNQNNF aggregates has been performed to analyze the behavior of the assemblies in a non-crystalline context. Stability, dynamics, and structural features suggest that SNQNNF assemblies are very good candidates to be involved in the structure of PrP fibrils. In addition, the analysis of small aggregates shows that steric zipper interfaces are able to stabilize assemblies composed of four strands per sheet. Altogether, the present findings indicate that steric zipper may play a key role in prion diseases. This suggestion is also corroborated by MD analyses of point mutations within the region 170-175.     

The role of the 132-160 region in prion protein conformational transitions

The native conformation of host-encoded cellular prion protein (PrP(C)) is metastable. As a result of a post-translational event, PrP(C) can convert to the scrapie form (PrP(Sc)), which emerges as the essential constituent of infectious prions. Despite thorough research, the mechanism underlying this conformational transition remains unknown. However, several studies have highlighted the importance of the N-terminal region spanning residues 90-154 in PrP folding. In order to understand why PrP folds into two different conformational states exhibiting distinct secondary and tertiary structure, and to gain insight into the involvement of this particular region in PrP transconformation, we studied the pressure-induced unfolding/ refolding of recombinant Syrian hamster PrP expanding from residues 90-231, and compared it with heat unfolding. By using two intrinsic fluorescent variants of this protein (Y150W and F141W), conformational changes confined to the 132-160 segment were monitored. Multiple conformational states of the Trp variants, characterized by their spectroscopic properties (fluorescence and UV absorbance in the fourth derivative mode), were achieved by tuning the experimental conditions of pressure and temperature. Further insight into unexplored conformational states of the prion protein, likely to mimic the in vivo structural change, was obtained from pressure-assisted cold unfolding. Furthermore, salt-induced conformational changes suggested a structural stabilizing role of Tyr150 and Phe141 residues, slowing down the conversion to a beta-sheet form.     

Implications of prion adaptation and evolution paradigm for human neurodegenerative diseases

There is a growing body of evidence indicating that number of human neurodegenerative diseases, including Alzheimer disease, Parkinson disease, fronto-temporal dementias, and amyotrophic lateral sclerosis, propagate in the brain via prion-like intercellular induction of protein misfolding. Prions cause lethal neurodegenerative diseases in humans, the most prevalent being sporadic Creutzfeldt-Jakob disease (sCJD); they self-replicate and spread by converting the cellular form of prion protein (PrP^C) to a misfolded pathogenic conformer (PrP^Sc). The extensive phenotypic heterogeneity of human prion diseases is determined by polymorphisms in the prion protein gene, and by prion strain-specific conformation of PrP^Sc. Remarkably, even though informative nucleic acid is absent, prions may undergo rapid adaptation and evolution in cloned cells and upon crossing the species barrier. In the course of our investigation of this process, we isolated distinct populations of PrP^Sc particles that frequently co-exist in sCJD. The human prion particles replicate independently and undergo competitive selection of those with lower initial conformational stability. Exposed to mutant substrate, the winning PrP^Sc conformers are subject to further evolution by natural selection of the subpopulation with the highest replication rate due to the lowest stability. Thus, the evolution and adaptation of human prions is enabled by a dynamic collection of distinct populations of particles, whose evolution is governed by the selection of progressively less stable, faster replicating PrP^Sc conformers. This fundamental biological mechanism may explain the drug resistance that some prions gained after exposure to compounds targeting PrP^Sc. Whether the phenotypic heterogeneity of other neurodegenerative diseases caused by protein misfolding is determined by the spectrum of misfolded conformers (strains) remains to be established. However, the prospect that these conformers may evolve and adapt by a prion-like mechanism calls for the reevaluation of therapeutic strategies that target aggregates of misfolded proteins, and argues for new therapeutic approaches that will focus on prior pathogenetic steps.     

Conformational stability of PrP amyloid fibrils controls their smallest possible fragment size

Fibril fragmentation is considered to be an essential step in prion replication. Recent studies have revealed a strong correlation between the incubation period to prion disease and conformational stability of synthetic prions. To gain insight into the molecular mechanism that accounts for this correlation, we proposed that the conformational stability of prion fibrils controls their intrinsic fragility or the size of the smallest possible fibrillar fragments. Using amyloid fibrils produced from full-length mammalian prion protein under three growth conditions, we found a correlation between conformational stability and the smallest possible fragment sizes. Specifically, the fibrils that were conformationally less stable were found to produce shorter pieces upon fragmentation. Site-specific denaturation experiments revealed that the fibril conformational stability was controlled by the region that acquires a cross-β-sheet structure. Using atomic force microscopy imaging, we found that fibril fragmentation occurred in both directions—perpendicular to and along the fibrillar axis. Two mechanisms of fibril fragmentation were identified: (i) fragmentation caused by small heat shock proteins, including αB-crystallin, and (ii) fragmentation due to mechanical stress arising from adhesion of the fibril to a surface. This study provides new mechanistic insight into the prion replication mechanism and offers a plausible explanation for the correlation between conformational stability of synthetic prions and incubation time to prion disease.     

Two-rung model of a left-handed beta-helix for prions explains species barrier and strain variation in transmissible spongiform encephalopathies

In this study, a new beta-helical model is proposed that explains the species barrier and strain variation in transmissible spongiform encephalopathies. The left-handed beta-helix serves as a structural model that can explain the seeded growth characteristics of beta-sheet structure in PrP(Sc) fibrils. Molecular dynamics simulations demonstrate that the left-handed beta-helix is structurally more stable than the right-handed beta-helix, with a higher beta-sheet content during the simulation and a better distributed network of inter-strand backbone-backbone hydrogen bonds between parallel beta-strands of different rungs. Multiple sequence alignments and homology modelling of prion sequences with different rungs of left-handed beta-helices illustrate that the PrP region with the highest beta-helical propensity (residues 105-143) can fold in just two rungs of a left-handed beta-helix. Even if no other flanking sequence participates in the beta-helix, the two rungs of a beta-helix can give the growing fibril enough elevation to accommodate the rest of the PrP protein in a tight packing at the periphery of a trimeric beta-helix. The folding of beta-helices is driven by backbone-backbone hydrogen bonding and stacking of side-chains in adjacent rungs. The sequence and structure of the last rung at the fibril end with unprotected beta-sheet edges selects the sequence of a complementary rung and dictates the folding of the new rung with optimal backbone hydrogen bonding and side-chain stacking. An important side-chain stack that facilitates the beta-helical folding is between methionine residues 109 and 129, which explains their importance in the species barrier of prions. Because the PrP sequence is not evolutionarily optimised to fold in a beta-helix, and because the beta-helical fold shows very little sequence preference, alternative alignments are possible that result in a different rung able to select for an alternative complementary rung. A different top rung results in a new strain with different growth characteristics. Hence, in the present model, sequence variation and alternative alignments clarify the basis of the species barrier and strain specificity in PrP-based diseases.