Real-time kinetics of discontinuous and highly conformational metal-ion binding sites of prion protein

The prion protein (PrP) is a metalloprotein with an unstructured region covering residues 60–91 that bind two to six Cu(II) ions cooperatively. Cu can bind to PrP regions C-terminally to the octarepeat region involving residues His111 and/or His96. In addition to Cu(II), PrP binds Zn(II), Mn(II) and Ni(II) with binding constants several orders of magnitudes lower than those determined for Cu. We used for the first time surface plasmon resonance (SPR) analysis to dissect metal binding to specific sites of PrP domains and to determine binding kinetics in real time. A biosensor assay was established to measure the binding of PrP-derived synthetic peptides and recombinant PrP to nitrilotriacetic acid chelated divalent metal ions. We have identified two separate binding regions for binding of Cu to PrP by SPR, one in the octarepeat region and the second provided by His96 and His111, of which His96 is more essential for Cu coordination. The octarepeat region at the N-terminus of PrP increases the affinity for Cu of the full-length protein by a factor of 2, indicating a cooperative effect. Since none of the synthetic peptides covering the octarepeat region bound to Mn and recombinant PrP lacking this sequence were able to bind Mn, we propose a conformational binding site for Mn involving residues 91–230. A novel low-affinity binding site for Co(II) was discovered between PrP residues 104 and 114, with residue His111 being the key amino acid for coordinating Co(II). His111 is essential for Co(II) binding, whereas His96 is more important than His111 for binding of Cu(II).     

Antibodies to a Nonconjugated Prion Protein Peptide 95-123 Interfere with PrP<Superscript><Emphasis Type="Bold">Sc</Emphasis></Superscript> Propagation in Prion-Infected Cells

Summary 1. Vaccination-induced anti-prion protein antibodies are presently regarded as a promising approach toward treatment of prion diseases. Here, we investigated the ability of five peptides corresponding to three different regions of the bovine prion protein (PrP) to elicit antibodies interfering with PrP^Sc propagation in prion-infected cells. 2. Rabbits were immunized with free nonconjugated peptides. Obtained immune sera were tested in enzyme-linked immunosorbent assay (ELISA) and immunoblot for their binding to recombinant PrP and cell-derived pathogenic isoform (PrP^Sc) and normal prion protein (PrP^c), respectively. Sera positive in all tests were chosen for PrP^Sc inhibition studies in cell culture. 3. All peptides induced anti-peptide antibodies, most of them reacting with recombinant PrP. Moreover, addition of the serum specific to peptide 95–123 led to a transient reduction of PrP^Sc levels in persistently prion-infected cells. 4. Thus, anti-PrP antibodies interfering with PrP^Sc propagation were induced with a prion protein peptide nonconjugated to a protein carrier. These results point to the potential application of the nonconjugated peptide 95–123 for the treatment of prion diseases.     

Disulfide bond as a structural determinant of prion protein membrane insertion

Conversion of the normal soluble form of prion protein, PrP (PrP^C), to proteinase K-resistant form (PrP^Sc) is a common molecular etiology of prion diseases. Proteinase K-resistance is attributed to a drastic conformational change from α-helix to β-sheet and subsequent fibril formation. Compelling evidence suggests that membranes play a role in the conformational conversion of PrP. However, biophysical mechanisms underlying the conformational changes of PrP and membrane binding are still elusive. Recently, we demonstrated that the putative transmembrane domain (TMD; residues 111–135) of Syrian hamster PrP penetrates into the membrane upon the reduction of the conserved disulfide bond of PrP. To understand the mechanism underlying the membrane insertion of the TMD, here we explored changes in conformation and membrane binding abilities of PrP using wild type and cysteine-free mutant. We show that the reduction of the disulfide bond of PrP removes motional restriction of the TMD, which might, in turn, expose the TMD into solvent. The released TMD then penetrates into the membrane. We suggest that the disulfide bond regulates the membrane binding mode of PrP by controlling the motional freedom of the TMD.     

Cross interaction of β-amyloid peptide and prion protein fragments

Summary This article presents kinetic studies of cross interaction of β-amyloid peptide and prion protein fragments. Syntheses of three peptides (β25-35, β22-35 and PrP 109–126) were performed. Those peptides were used for aggregation studies in PBS and TRIS buffers using HPLC with DAD detector. Comparison of aggregation of peptides alone and in combination with other fragments was investigated. In all cases aggregation was faster in PBS than in TRIS solution. Obtained results suggest that β-amyloid peptide and prion protein may interact to form macromolecular complexes with different ability for aggregation.     

The synthesis and spectroscopic analysis of the neurotoxic prion peptide 106-126: Comparative use of manual Boc and Fmoc chemistry

Summary A peptide corresponding to residues 106–126 of the human prion protein (PrP) possesses the neurotoxic and amyloidogenic properties of the infectious form of the parental protein. This peptide is now identified as a ‘difficult sequence’ and synthesis using conventional manual Fmoc chemistry was unsuccessful with acylation terminating at a central core of hydrophobic amino acids. The use of tetramethylfluoroformamidinium hexafluorophosphate and 1-methyl-2-pyrrolidone as anti-aggregatory agents in the coupling steps improved the synthesis but still resulted in an incomplete peptide. The incorporation ofN-(2-hydroxy-4-methoxybenzyl)protection at glycine residues 119 and 124 enabled synthesis of the full length peptide in low yield. Synthesis using Boc chemistry within situ neutralisation gave the full length peptide in high yield.     

Probing copper2+ binding to the prion protein using diamagnetic nickel2+ and 1H NMR: the unstructured N terminus facilitates the coordination of six copper2+ ions at physiological concentrations

The prion protein (PrP) is a Cu2+ binding cell surface glyco-protein. Misfolding of PrP into a beta-sheet rich conformation is associated with transmissible spongiform encephalopathies. Here we use Ni2+ as a diamagnetic probe to further understand Cu2+ binding to PrP. Like Cu2+, Ni2+ preferentially binds to an unstructured region between residues 90 and 126 of PrP, which is a key region for amyloidogenicity and prion propagation. Using both 1H NMR and visible-circular dichroism (CD) spectroscopy, we show that two Ni2+ ions bind to His96 and His111 independently of each other. 1H NMR indicates that both Ni2+ binding sites form square-planar diamagnetic complexes. We have previously shown that Cu2+ forms a paramagnetic square-planar complex in this region, suggesting that Ni2+ could be used as a probe for Cu2+ binding. In addition, competition studies show that two Cu2+ ions can displace Ni2+ from these sites. Upon Ni2+ addition 1H NMR changes in chemical shifts indicate the imidazole ring and amide nitrogen atoms to the N terminus of both His96 and His111 act as coordinating ligands. Use of peptide fragments confirm that PrP(92-96) and PrP(107-111) represent the minimal binding motif for the two Ni2+ binding sites. Analysis of Cu2+ loaded visible-CD spectra show that as with Ni2+, PrP(90-115) binds two Cu2+ ions at His96 and His111 independently of each other. Visible CD studies with PrP(23-231Delta51-90), a construct of PrP(23-231) with the octarepeat region deleted to improve solubility, confirm binding of Ni2+ to His96 and His111 in octarepeat deleted PrP(23-231). The structure of the Cu/Ni complexes is discussed in terms of the implications for prion protein function and disease.     

The role of prion peptide structure and aggregation in toxicity and membrane binding

Prion diseases are neurodegenerative disorders associated with a conformational change in the normal cellular isoform of the prion protein, PrP(C), to an abnormal scrapie isoform, PrP(SC). Unlike the alpha-helical PrP(C), the protease-resistant core of PrP(SC) is predominantly beta-sheet and possesses a tendency to polymerize into amyloid fibrils. We performed experiments with two synthetic human prion peptides, PrP(106-126) and PrP(127-147), to determine how peptide structure affects neurotoxicity and protein-membrane interactions. Peptide solutions possessing beta-sheet and amyloid structures were neurotoxic to PC12 cells in vitro and bound with measurable affinities to cholesterol-rich phospholipid membranes at ambient conditions, but peptide solutions lacking stable beta-sheet structures and amyloid content were nontoxic and possessed less than one tenth of the binding affinities of the amyloid-containing peptides. Regardless of structure, the peptide binding affinities to cholesterol-depleted membranes were greatly reduced. These results suggest that the beta-sheet and amyloid structures of the prion peptides give rise to their toxicity and membrane binding affinities and that membrane binding affinity, especially in cholesterol-rich environments, may be related to toxicity. Our results may have significance in understanding the role of the fibrillogenic cerebral deposits associated with some of the prion diseases in neurodegeneration and may have implications for other amyloidoses.     

Identification of an epitope in the C terminus of normal prion protein whose expression is modulated by binding events in the N terminus

We have characterized the epitopes of a panel of 12 monoclonal antibodies (Mabs) directed to normal human cellular prion protein (PrP(C)) using ELISA and Western blotting of recombinant PrP or synthetic peptide fragments of PrP. The first group of antibodies, which is represented by Mabs 5B2 and 8B4, reacts with PrP(23-145), indicating that the epitopes for these Mabs are located in the 23 to 145 N-terminal region of human PrP. The second group includes Mabs 1A1, 6H3, 7A9, 8C6, 8H4, 9H7 and 2G8. These antibodies bind to epitopes localized within N-terminally truncated recombinant PrP(90-231). Finally, Mabs 5C3, 2C9 and 7A12 recognize both PrP(23-145) and PrP(90-231), suggesting that the epitopes for this group are located in the region encompassing residues 90 to 145. By Western blotting with PepSpot(TM), only three of Mabs studied (5B2, 8B4 and 2G8) bind to linear epitopes that are present in 13-residue long synthetic peptides corresponding to human PrP fragments. The remaining nine Mabs appear to recognize conformational epitopes. Two N terminus-specific Mabs were found to prevent the binding of the C terminus-specific Mab 6H3. This observation suggests that the unstructured N-terminal region may influence the local conformation within the folded C-terminal domain of prion protein.     

羊朊毒体单抗结合表位分析

通过分段表达PrP核心片段和人工合成多肽,分析5株羊朊毒体单抗结合表位。分段表达PrP核心片段,通过PCR方法扩增目的片段,经酶切、连接后,将目的片段插入质粒pET32a,在大肠杆菌BL21中表达。将表达的系列融合蛋白与单抗进行免疫转印试验,根据反应情况确定单抗结合的大致部位,在此基础上设计合成多条针对性多肽,用ELISA方法进一步确定3株单抗的结合部位;通过与6段融合蛋白反应证明5株单抗的结合部位分别为:2H3在199aa～213aa之间,4C6、5F11和7F11在139aa～168aa之间,7F1在214aa～227aa之间,与3段人工合成多肽进行ELISA反应进一步得到4C6、5F11和7F11抗原结合表位在149aa～158aa之间;本研究确定了5株单抗在PrP分子上的结合部位,为羊痒病和牛海绵状脑病的检测、发病机制的研究奠定了基础。     

The role of hydrophobic interactions in amyloidogenesis: example of prion-related polypeptides

Conversion of the non-infectious, cellular form of the prion protein (PrP(C)) to the infectious form (PrP(Sc)) is thought to be driven by an alpha-helical to beta-sheet conformational transition. To reveal the sequence determinants which encourage the transition to beta-fold, we study the synthetic peptides associated with hydrophobic conserved fragments of the N-terminal region of the prion protein. The structure of peptides in solution was probed under various thermodynamic conditions employing circular dichroism and steady state fluorescence spectroscopy as well as dye binding assays. The fluorescence methods utilized pyrene residues covalently attached to the end of the model peptides. In aqueous solutions, the structure assessments indicate the formation of metastable peptide aggregates; the molecular conformations within the peptide micelles are largely coiled. This stage in molecular assembly exists without significant beta-strand formation, i.e., before the appearance of any ordered secondary structure detectable by circular dichroism. At moderate concentrations of trifluoroethanol and/or acetonitrile, the conformational ensemble shifts towards beta-strand formation, and the population of the amorphous aggregates decreases significantly. Overall, the present data indicate that hydrophobic interactions between side chains of the peptide variants prevent, in fact, the formation of the rigid beta-sheet structures. Encouragement of beta-folds requires the destabilization of local interactions in the peptide chain, which in vivo might be possible within cell membranes as well as within partly folded molecular forms.     

Difference in redox behaviors between copper-binding octarepeat and nonoctarepeat sites in prion protein

We studied the redox behavior of copper-binding sites in prion protein (PrP) to clarify copper’s role in the pathological mechanism underlying prion diseases. We investigated the coordination structures, binding affinities, and redox potentials of copper-binding peptide fragments derived from the N-terminal domain of PrP by density functional theory calculations. We used four models for copper-binding moieties in PrP(60–96): two were derived from the PHGGGWGQ octapeptide repeat region of PrP(60–91), and the others were tripeptide Gly-Thr-His fragments derived from the copper-binding nonoctarepeat site around His96. We found that such PrP-derived copper-binding complexes exhibit conformationally dependent redox behavior; for example, the copper-binding complex derived from the octarepeat region tends to possess high reduction potential for the Cu(II)/Cu(I) couple, exceeding 0 V versus the standard hydrogen electrode, whereas the copper-binding nonoctarepeat model around His96 tends to possess high oxidation potential for the Cu(II)/Cu(III) couple and stabilize the higher-valent Cu(III) state. It is possible that such distinct redox activities of a copper-binding PrP are involved in the mechanism underlying prion diseases.     

Human collagen II peptide 256–271 preferentially binds to HLA-DR molecules associated with susceptibility to rheumatoid arthritis

 The binding ability of 23 overlapping peptides, all derived from the CB11 fragment of CII, was tested on several HLA-DR molecules associated or not with disease susceptibility. These experiments were performed on a variety of cells expressing different HLA-DR molecules, using both indirect and direct binding assays. The CII (256–271) fragment was shown to bind to a restricted population among which the HLA-DR molecules associated with susceptibility to rheumatoid arthritis. The results also clearly indicate that the binding specificity of CII (256–271), among the DR4 molecules, is controlled by the nature of the HLA-DR molecule β-chain residues 71 and 74, residues previously shown by X-ray crystallography to be involved in the HLA-DR/peptide interaction. The human CII (256–271) peptide is thus likely to play a role in the disease process. Received: 6 May 1998 / Revised: 16 July 1998     

Prion protein region 23–32 interacts with tubulin and inhibits microtubule assembly

In previous studies we have demonstrated that prion protein (PrP) binds directly to tubulin and this interaction leads to the inhibition of microtubule formation by inducement of tubulin oligomerization. This report is aimed at mapping the regions of PrP and tubulin involved in the interaction and identification of PrP domains responsible for tubulin oligomerization. Preliminary studies focused our attention to the N‐terminal flexible part of PrP encompassing residues 23–110. Using a panel of deletion mutants of PrP, we identified two microtubule‐binding motifs at both ends of this part of the molecule. We found that residues 23–32 constitute a major site of interaction, whereas residues 101–110 represent a weak binding site. The crucial role of the 23–32 sequence in the interaction with tubulin was confirmed employing chymotryptic fragments of PrP. Surprisingly, the octarepeat region linking the above motifs plays only a supporting role in the interaction. The binding of Cu²⁺ to PrP did not affect the interaction. We also demonstrate that PrP deletion mutants lacking residues 23–32 exhibit very low efficiency in the inducement of tubulin oligomerization. Moreover, a synthetic peptide corresponding to this sequence, but not that identical with fragment 101–110, mimics the effects of the full‐length protein on tubulin oligomerization and microtubule assembly. At the cellular level, peptide composed of the PrP motive 23–30 and signal sequence (1–22) disrupted the microtubular cytoskeleton. Using tryptic and chymotryptic fragments of α‐ and β‐tubulin, we mapped the docking sites for PrP within the C‐terminal domains constituting the outer surface of microtubule. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Inhibition of interactions and interconversions of prion protein isoforms by peptide fragments from the C-terminal folded domain

The formation of protease-resistant prion protein (PrP-res or PrP(Sc)) involves selective interactions between PrP-res and its normal protease-sensitive counterpart, PrP-sen or PrP(C). Previous studies have shown that synthetic peptide fragments of the PrP sequence corresponding to residues 119-136 of hamster PrP (Ha119-136) can selectively block PrP-res formation in cell-free systems and scrapie-infected tissue culture cells. Here we show that two other peptides corresponding to residues 166-179 (Ha166-179) and 200-223 (Ha200-223) also potently inhibit the PrP-res induced cell-free conversion of PrP-sen to the protease-resistant state. In contrast, Ha121-141, Ha180-199, and Ha218-232 were much less effective as inhibitors. Mechanistic analyses indicated that Ha166-179, Ha200-223, and peptides containing residues 119-136 inhibit primarily by binding to PrP-sen and blocking its binding to PrP-res. Circular dichroism analyses indicated that Ha117-141 and Ha200-223, but not non-inhibitory peptides, readily formed high beta-sheet structures when placed under the conditions of the conversion reaction. We conclude that these inhibitory peptides may mimic contact surfaces between PrP-res and PrP-sen and thereby serve as models of potential therapeutic agents for transmissible spongiform encephalopathies.     

Interaction between Human Prion Protein and Amyloid-β (Aβ) Oligomers: ROLE OF N-TERMINAL RESIDUES*

Soluble oligomers of Aβ42 peptide are believed to play a major role in the pathogenesis of Alzheimer disease (AD). It was recently found that at least some of the neurotoxic effects of these oligomers may be mediated by specific binding to the prion protein, PrP^C, on the cell surface (Laurén, J., Gimbel, D. A., Nygaard, H. B., Gilbert, J. W., and Strittmatter, S. M. (2009) Nature 457, 1128–1132). Here we characterized the interaction between synthetic Aβ42 oligomers and the recombinant human prion protein (PrP) using two biophysical techniques: site-directed spin labeling and surface plasmon resonance. Our data indicate that this binding is highly specific for a particular conformation adopted by the peptide in soluble oligomeric species. The binding appears to be essentially identical for the Met¹²⁹ and Val¹²⁹ polymorphic forms of human PrP, suggesting that the role of PrP codon 129 polymorphism as a risk factor in AD is due to factors unrelated to the interaction with Aβ oligomers. It was also found that in addition to the previously identified ∼95–110 segment, the second region of critical importance for the interaction with Aβ42 oligomers is a cluster of basic residues at the extreme N terminus of PrP (residues 23–27). The deletion of any of these segments results in a major loss of the binding function, indicating that these two regions likely act in concert to provide a high affinity binding site for Aβ42 oligomers. This insight may help explain the interplay between the postulated protective and pathogenic roles of PrP in AD and may contribute to the development of novel therapeutic strategies as well.     

Toward molecular dissection of PrPC-PrPSc interactions

Direct interaction between endogenous cellular prion protein (PrP(C)) and misfolded, disease-associated (PrP(Sc)) conformers is a key event in prion propagation, which precedes templated conversion of PrP(C) into nascent PrP(Sc) and prion infectivity. Although almost none of the molecular details of this pivotal process are understood, the persistence of individual prion strains suggests that assembly of the prion replicative complex is mechanistically precise. To systematically map defined regions of PrP(C) sequence that bind tightly to PrP(Sc), we have generated a comprehensive panel of over 45 motif-grafted antibodies containing overlapping peptide grafts collectively spanning PrP residues 19-231. Grafted antibody binding experiments, performed under stringent conditions, clearly identified only three distinct and independent high affinity PrP(Sc) recognition motifs. The first of these binding motifs lies at the very N-terminal region of the mature PrP molecule within PrP-(23-33); the second motif lies within PrP-(98-110); and the third is contained within PrP-(136-158). Mutational analyses of these PrP(Sc)-binding regions revealed that reactivity of the 23-33 and 98-110 segments are largely dependent upon the presence of multiple positively charged amino acid residues. These studies yield new insight into critical peptidic components composing one side of the prion replicative interface.     

Heat shock protein 104 inhibited the fibrillization of prion peptide 106-126 and disassembled prion peptide 106-126 fibrils in vitro

Amyloid-like fibrils have been associated with the pathogenesis of human prion diseases. Prion peptide of aa 106-126 (PrP106-126) exhibits many PrP(Sc)-like biochemical features, forming amyloid-like fibrils in vitro. Here, we found that the recombinant yeast-derived molecular chaperon Hsp104 inhibited significantly the fibril assembly of the synthetic PrP106-126 peptide by dynamic ThT assays in vitro. EM assays revealed almost no fibril-like structure after incubation of the synthetic PrP106-126 peptides with Hsp104 for 12h. Circular dichroism assays identified that treatment of Hsp104 shifted the secondary structure of PrP106-126 fibrils from β-sheet to a random coil. MTT tests confirmed that interaction of PrP106-126 with Hsp104 maintained the toxicity of PrP106-126 on human neuroblastoma cell line SK-N-SH. Additionally, Hsp104 was able to disassemble the mature PrP106-126 fibrils in vitro, leading to recovering the cytotoxicity of PrP106-126 on SK-N-SH cells. Our study provides the molecular evidences that the yeast-derived Hsp104 can interfere in the fibril assembly and disassembly of human PrP106-126 segment.     

The binding of the molecular chaperone Hsc70 to the prion protein PrP is modulated by pH and copper

Conformational transitions in the prion protein (PrP) are thought to be central to the pathogenesis of the transmissible spongiform encephalopathies (TSE), such as Creutzfeldt-Jacob disease and bovine spongiform encephalopathy. Studies of prion phenomena in yeast have shown that molecular chaperones play an important role in prion related conformational transitions. Here, we investigated the interaction of the molecular chaperone Hsc70 (HSPA8) with recombinant PrP in vitro using an ELISA based assay. Hsc70 bound to PrP in a saturable manner over a range of temperatures and binding was greatest at low pH. Surprisingly, Hsc70 bound more avidly to native recombinant PrP than to denatured PrP or other potential clients, such as denatured luciferase or rhodanese. Hsc70 binding to native PrP was enhanced by incubation with Cu²⁺ at low pH. The Hsc70 binding sites in PrP were analysed using a synthetic PrP-derived peptide array. The binding of Hsc70 to PrP was reminiscent of the published ovine PrP to bovine PrP binding data and included two potential regions of binding that correspond to the proposed ‘protein X’ binding sites in PrP. Synthetic peptides corresponding to these sites specifically inhibited the Hsc70 interaction with native PrP, further demonstrating that Hsc70 might interact with PrP via this epitope. The data suggest that molecular chaperones could modulate important PrP conformational transitions or protein–protein interactions in TSE pathogenesis.     

Structural Requirements of Maxadilan,a Non-Mammalian Potent Vasodilatory Peptide,for Interaction with the PAC-1 Receptor from Rat Brain Using a Synthetic Mini-Library

Although there is no amino acid sequence similarity between maxadilan (Maxa) and pituitary adenylate cyclase activating polypeptide (PACAP), our synthetic Maxa was found to bind PACAP specific receptors (PAC-1 receptors) with a high affinity, but low potency for the accumulation of cAMP in PC12 cells. Competitive binding studies of ¹²⁵I-PACAP-27 to rat cortical membranes allowed exploration of the structural requirements for this interaction using mini-libraries constructed by solid-phase peptided synthesis, that include disulfide isomers, N-, C- and middle segment deleted peptides and analogs. Maxa as well as PACAP38 inhibited the specific binding of ¹²⁵I-PACAP-27 with IC₅₀ values of 3.89 and 4.90 nM, respectively. The most potent derivative of our synthetic Maxa-analogs with an IC₅₀ value of 1.99 nM was Maxa[1–23 + 43–61, S–S^14–51 Ala^1,5] which consists of N- (position 1–23) and C- (position 43-61) terminal linear fragments cross-linked by a disulfide bridge between positions 14 and 51. This peptide did not increase intracellular cAMP, at a concentration of 100 nM, but inhibited cAMP accumulation induced by 1 nM PACAP-27 in PC12 cells, whereas wild Maxa increased intracellular cAMP although it was weaker than PACAP-27. Our data suggest deletion of the middle segment between residues 24–42 affords some derivatives that behave as low affinity antagonists. This paper is dedicated to the memory of Professor Bruce Merrifield, a pioneer and one of the most important contributors to solid-phase synthesis.     

Synthetic miniprion PrP106

Elucidation of structure and biological properties of the prion protein scrapie (PrP(Sc)) is fundamental to an understanding of the mechanism of conformational transition of cellular (PrP(C)) into disease-specific isoforms and the pathogenesis of prion diseases. Unfortunately, the insolubility and heterogeneity of PrP(Sc) have limited these studies. The observation that a construct of 106 amino acids (termed PrP106 or miniprion), derived from mouse PrP and containing two deletions (Delta 23-88, Delta 141-176), becomes protease-resistant when expressed in scrapie-infected neuroblastoma cells and sustains prion replication when expressed in PrP(0/0) mice prompted us to generate a corresponding synthetic peptide (sPrP106) to be used for biochemical and cell culture studies. sPrP106 was obtained successfully with a straightforward procedure, which combines classical stepwise solid phase synthesis with a purification strategy based on transient labeling with a lipophilic chromatographic probe. sPrP106 readily adopted a beta-sheet structure, aggregated into branched filamentous structures without ultrastructural and tinctorial properties of amyloid, exhibited a proteinase K-resistant domain spanning residues 134-217, was highly toxic to primary neuronal cultures, and induced a remarkable increase in membrane microviscosity. These features are central properties of PrP(Sc) and make sPrP106 an excellent tool for investigating the molecular basis of the conformational conversion of PrP(C) into PrP(Sc) and prion disease pathogenesis.