Globular domain of the prion protein needs to be unlocked by domain swapping to support prion protein conversion

Prion diseases are fatal transmissible neurodegenerative diseases affecting many mammalian species. The normal prion protein (PrP) converts into a pathological aggregated form, PrPSc, which is enriched in the β-sheet structure. Although the high resolution structure of the normal PrP was determined, the structure of the converted form of PrP remains inaccessible to high resolution techniques. To map the PrP conversion process we introduced disulfide bridges into different positions within the globular domain of PrP, tethering selected secondary structure elements. The majority of tethered PrP mutants exhibited increased thermodynamic stability, nevertheless, they converted efficiently. Only the disulfides that tether subdomain B1-H1-B2 to subdomain H2-H3 prevented PrP conversion in vitro and in prion-infected cell cultures. Reduction of disulfides recovered the ability of these mutants to convert, demonstrating that the separation of subdomains is an essential step in conversion. Formation of disulfide-linked proteinase K-resistant dimers in fibrils composed of a pair of single cysteine mutants supports the model based on domain-swapped dimers as the building blocks of prion fibrils. In contrast to previously proposed structural models of PrPSc suggesting conversion of large secondary structural segments, we provide evidence for the conservation of secondary structural elements of the globular domain upon PrP conversion. Previous studies already showed that dimerization is the rate-limiting step in PrP conversion. We show that separation and swapping of subdomains of the globular domain is necessary for conversion. Therefore, we propose that the domain-swapped dimer of PrP precedes amyloid formation and represents a potential target for therapeutic intervention.     

Structural polymorphism in amyloids: new insights from studies with Y145Stop prion protein fibrils

The C-terminally-truncated human prion protein variant Y145Stop (or PrP23-144), associated with a familial prion disease, provides a valuable model for studying the fundamental properties of protein amyloids. In previous solid-state NMR experiments, we established that the β-sheet core of the PrP23-144 amyloid is composed of two β-strand regions encompassing residues ～113-125 and ～130-140. The former segment contains a highly conserved hydrophobic palindrome sequence, (113)AGAAAAGA(120), which has been considered essential to PrP conformational conversion. Here, we examine the role of this segment in fibrillization of PrP23-144 using a deletion variant, Δ113-120 PrP23-144, in which the palindrome sequence is missing. Surprisingly, we find that deletion of the palindrome sequence affects neither the amyloidogenicity nor the polymerization kinetics of PrP23-144, although it does alter amyloid conformation and morphology. Using two-dimensional and three-dimensional solid-state NMR methods, we find that Δ113-120 PrP23-144 fibrils contain an altered β-core extended N-terminally to residue ～106, encompassing residues not present in the core of wild-type PrP23-144 fibrils. The C-terminal β-strand of the core, however, is similar in both fibril types. Collectively, these data indicate that amyloid cores of PrP23-144 variants contain "essential" (i.e. nucleation-determining) and "nonessential" regions, with the latter being "movable" in amino acid sequence space. These findings reveal an intriguing new mechanism for structural polymorphism in amyloids and suggest a potential means for modulating the physicochemical properties of amyloid fibrils without compromising their polymerization characteristics.     

Interactions between the conserved hydrophobic region of the prion protein and dodecylphosphocholine micelles

The three-dimensional structure of PrP110-136, a peptide encompassing the conserved hydrophobic region of the human prion protein, has been determined at high resolution in dodecylphosphocholine micelles by NMR. The results support the conclusion that the (Ctm)PrP, a transmembrane form of the prion protein, adopts a different conformation than the reported structures of the normal prion protein determined in solution. Paramagnetic relaxation enhancement studies with gadolinium-diethylenetriaminepentaacetic acid indicated that the conserved hydrophobic region peptide is not inserted symmetrically in the micelle, thus suggesting the presence of a guanidium-phosphate ion pair involving the side chain of the terminal arginine and the detergent headgroup. Titration of dodecylphosphocholine into a solution of PrP110-136 revealed the presence of a surface-bound species. In addition, paramagnetic probes located the surface-bound peptide somewhere below the micelle-water interface when using the inserted helix as a positional reference. This localization of the unknown population would allow a similar ion pair interaction.     

Dual conformation of H2H3 domain of prion protein in mammalian cells

The concept of prion is applied to protein modules that share the ability to switch between at least two conformational states and transmit one of these through intermolecular interaction and change of conformation. Although much progress has been achieved through the understanding of prions from organisms such as Saccharomyces cerevisiae, Podospora anserina, or Aplysia californica, the criteria that qualify a protein module as a prion are still unclear. In addition, the functionality of known prion domains fails to provide clues to understand the first identified prion, the mammalian infectious prion protein, PrP. To address these issues, we generated mammalian cellular models of expression of the C-terminal two helices of PrP, H2 and H3, which have been hypothesized, among other models, to hold the replication and conversion properties of the infectious PrP. We found that the H2H3 domain is an independent folding unit that undergoes glycosylations and glycosylphosphatidylinositol anchoring similar to full-length PrP. Surprisingly, in some conditions the normally folded H2H3 was able to systematically go through a conversion process and generate insoluble proteinase K-resistant aggregates. This structural switch involves the assembly of amyloid structures that bind thioflavin S and oligomers that are reactive to A11 antibody, which specifically detects protein oligomers from neurological disorders. Overall, we show that H2H3 is a conformational switch in a cellular context and is thus suggested to be a candidate for the conversion domain of PrP.     

Destruction of amyloid fibrils of keratoepithelin peptides by laser irradiation coupled with amyloid-specific thioflavin T

Mutations in keratoepithelin are associated with blinding ocular diseases, including lattice corneal dystrophy type 1 and granular corneal dystrophy type 2. These diseases are characterized by deposits of amyloid fibrils and/or granular non-amyloid aggregates in the cornea. Removing the deposits in the cornea is important for treatment. Previously, we reported the destruction of amyloid fibrils of β(2)-microglobulin K3 fragments and amyloid β by laser irradiation coupled with the binding of an amyloid-specific thioflavin T. Here, we studied the effects of this combination on the amyloid fibrils of two 22-residue fragments of keratoepithelin. The direct observation of individual amyloid fibrils was performed in real time using total internal reflection fluorescence microscopy. Both types of amyloid fibrils were broken up by the laser irradiation, dependent on the laser power. The results suggest the laser-induced destruction of amyloid fibrils to be a useful strategy for the treatment of these corneal dystrophies.     

The mechanism of fibril formation of a non-inhibitory serpin ovalbumin revealed by the identification of amyloidogenic core regions

Ovalbumin (OVA), a non-inhibitory member of the serpin superfamily, forms fibrillar aggregates upon heat-induced denaturation. Recent studies suggested that OVA fibrils are generated by a mechanism similar to that of amyloid fibril formation, which is distinct from polymerization mechanisms proposed for other serpins. In this study, we provide new insights into the mechanism of OVA fibril formation through identification of amyloidogenic core regions using synthetic peptide fragments, site-directed mutagenesis, and limited proteolysis. OVA possesses a single disulfide bond between Cys(73) and Cys(120) in the N-terminal helical region of the protein. Heat treatment of disulfide-reduced OVA resulted in the formation of long straight fibrils that are distinct from the semiflexible fibrils formed from OVA with an intact disulfide. Computer predictions suggest that helix B (hB) of the N-terminal region, strand 3A, and strands 4-5B are highly β-aggregation-prone regions. These predictions were confirmed by the fact that synthetic peptides corresponding to these regions formed amyloid fibrils. Site-directed mutagenesis of OVA indicated that V41A substitution in hB interfered with the formation of fibrils. Co-incubation of a soluble peptide fragment of hB with the disulfide-intact full-length OVA consistently promoted formation of long straight fibrils. In addition, the N-terminal helical region of the heat-induced fibril of OVA was protected from limited proteolysis. These results indicate that the heat-induced fibril formation of OVA occurs by a mechanism involving transformation of the N-terminal helical region of the protein to β-strands, thereby forming sequential intermolecular linkages.     

Amyloid fibrils of mammalian prion protein are highly toxic to cultured cells and primary neurons

A growing body of evidence indicates that small, soluble oligomeric species generated from a variety of proteins and peptides rather than mature amyloid fibrils are inherently highly cytotoxic. Here, we show for the first time that mature amyloid fibrils produced from full-length recombinant mammalian prion protein (rPrP) were highly toxic to cultured cells and primary hippocampal and cerebella neurons. Fibrils induced apoptotic cell death in a time- and dose-dependent manner. The toxic effect of fibrils was comparable with that exhibited by soluble small beta-oligomers generated from the same protein. Fibrils prepared from insulin were not toxic, suggesting that the toxic effect was not solely due to the highly polymeric nature of the fibrillar form. The cell death caused by rPrP fibrils or beta-oligomers was substantially reduced when expression of endogenous PrP(C) was down-regulated by small interfering RNAs. In opposition to the beta-oligomer and amyloid fibrils of rPrP, the monomeric alpha-helical form of rPrP stimulated neurite out-growth and survival of neurons. These studies illustrated that both soluble beta-oligomer and amyloid fibrils of the prion protein are intrinsically toxic and confirmed that endogenously expressed PrP(C) is required for mediating the toxicity of abnormally folded external PrP aggregates.     

Repeat domains of melanosome matrix protein Pmel17 orthologs form amyloid fibrils at the acidic melanosomal pH

Most amyloids are pathological, but fragments of Pmel17 form a functional amyloid in vertebrate melanosomes essential for melanin synthesis and deposition. We previously reported that only at the mildly acidic pH (4-5.5) typical of melanosomes, the repeat domain (RPT) of human Pmel17 can form amyloid in vitro. Combined with the known presence of RPT in the melanosome filaments and the requirement of this domain for filament formation, we proposed that RPT may be the core of the amyloid formed in vivo. Although most of Pmel17 is highly conserved across a broad range of vertebrates, the RPT domains vary dramatically, with no apparent homology in some cases. Here, we report that the RPT domains of mouse and zebrafish, as well as a small splice variant of human Pmel17, all form amyloid specifically at mildly acid pH (pH ～5.0). Protease digestion, mass per unit length measurements, and solid-state NMR experiments suggest that amyloid of the mouse RPT has an in-register parallel β-sheet architecture with two RPT molecules per layer, similar to amyloid of the Aβ peptide. Although there is no sequence conservation between human and zebrafish RPT, amyloid formation at acid pH is conserved.     

Conformational Stability of Mammalian Prion Protein Amyloid Fibrils Is Dictated by a Packing Polymorphism within the Core Region

Mammalian prion strains are believed to arise from the propagation of distinct conformations of the misfolded prion protein PrP^Sc. One key operational parameter used to define differences between strains has been conformational stability of PrP^Sc as defined by resistance to thermal and/or chemical denaturation. However, the structural basis of these stability differences is unknown. To bridge this gap, we have generated two strains of recombinant human prion protein amyloid fibrils that show dramatic differences in conformational stability and have characterized them by a number of biophysical methods. Backbone amide hydrogen/deuterium exchange experiments revealed that, in sharp contrast to previously studied strains of infectious amyloid formed from the yeast prion protein Sup35, differences in β-sheet core size do not underlie differences in conformational stability between strains of mammalian prion protein amyloid. Instead, these stability differences appear to be dictated by distinct packing arrangements (i.e. steric zipper interfaces) within the amyloid core, as indicated by distinct x-ray fiber diffraction patterns and large strain-dependent differences in hydrogen/deuterium exchange kinetics for histidine side chains within the core region. Although this study was limited to synthetic prion protein amyloid fibrils, a similar structural basis for strain-dependent conformational stability may apply to brain-derived PrP^Sc, especially because large strain-specific differences in PrP^Sc stability are often observed despite a similar size of the PrP^Sc core region.     

Generation of a new form of human PrP(Sc) in vitro by interspecies transmission from cervid prions

Prion diseases are infectious neurodegenerative disorders that affect humans and animals and that result from the conversion of normal prion protein (PrP(C)) into the misfolded prion protein (PrP(Sc)). Chronic wasting disease (CWD) is a prion disorder of increasing prevalence within the United States that affects a large population of wild and captive deer and elk. Determining the risk of transmission of CWD to humans is of utmost importance, considering that people can be infected by animal prions, resulting in new fatal diseases. To study the possibility that human PrP(C) can be converted into the misfolded form by CWD PrP(Sc), we performed experiments using the protein misfolding cyclic amplification technique, which mimics in vitro the process of prion replication. Our results show that cervid PrP(Sc) can induce the conversion of human PrP(C) but only after the CWD prion strain has been stabilized by successive passages in vitro or in vivo. Interestingly, the newly generated human PrP(Sc) exhibits a distinct biochemical pattern that differs from that of any of the currently known forms of human PrP(Sc). Our results also have profound implications for understanding the mechanisms of the prion species barrier and indicate that the transmission barrier is a dynamic process that depends on the strain and moreover the degree of adaptation of the strain. If our findings are corroborated by infectivity assays, they will imply that CWD prions have the potential to infect humans and that this ability progressively increases with CWD spreading.     

Mutant p53 Aggregates into Prion-like Amyloid Oligomers and Fibrils: IMPLICATIONS FOR CANCER

Over 50% of all human cancers lose p53 function. To evaluate the role of aggregation in cancer, we asked whether wild-type (WT) p53 and the hot-spot mutant R248Q could aggregate as amyloids under physiological conditions and whether the mutant could seed aggregation of the wild-type form. The central domains (p53C) of both constructs aggregated into a mixture of oligomers and fibrils. R248Q had a greater tendency to aggregate than WT p53. Full-length p53 aggregated into amyloid-like species that bound thioflavin T. The amyloid nature of the aggregates was demonstrated using x-ray diffraction, electron microscopy, FTIR, dynamic light scattering, cell viabilility assay, and anti-amyloid immunoassay. The x-ray diffraction pattern of the fibrillar aggregates was consistent with the typical conformation of cross β-sheet amyloid fibers with reflexions of 4.7 Å and 10 Å. A seed of R248Q p53C amyloid oligomers and fibrils accelerated the aggregation of WT p53C, a behavior typical of a prion. The R248Q mutant co-localized with amyloid-like species in a breast cancer sample, which further supported its prion-like effect. A tumor cell line containing mutant p53 also revealed massive aggregation of p53 in the nucleus. We conclude that aggregation of p53 into a mixture of oligomers and fibrils sequestrates the native protein into an inactive conformation that is typical of a prionoid. This prion-like behavior of oncogenic p53 mutants provides an explanation for the negative dominance effect and may serve as a potential target for cancer therapy.     

Amyloid-like fibrils from a domain-swapping protein feature a parallel, in-register conformation without native-like interactions

The formation of amyloid-like fibrils is characteristic of various diseases, but the underlying mechanism and the factors that determine whether, when, and how proteins form amyloid, remain uncertain. Certain mechanisms have been proposed based on the three-dimensional or runaway domain swapping, inspired by the fact that some proteins show an apparent correlation between the ability to form domain-swapped dimers and a tendency to form fibrillar aggregates. Intramolecular β-sheet contacts present in the monomeric state could constitute intermolecular β-sheets in the dimeric and fibrillar states. One example is an amyloid-forming mutant of the immunoglobulin binding domain B1 of streptococcal protein G, which in its native conformation consists of a four-stranded β-sheet and one α-helix. Under native conditions this mutant adopts a domain-swapped dimer, and it also forms amyloid-like fibrils, seemingly in correlation to its domain-swapping ability. We employ magic angle spinning solid-state NMR and other methods to examine key structural features of these fibrils. Our results reveal a highly rigid fibril structure that lacks mobile domains and indicate a parallel in-register β-sheet structure and a general loss of native conformation within the mature fibrils. This observation contrasts with predictions that native structure, and in particular intermolecular β-strand interactions seen in the dimeric state, may be preserved in "domain-swapping" fibrils. We discuss these observations in light of recent work on related amyloid-forming proteins that have been argued to follow similar mechanisms and how this may have implications for the role of domain-swapping propensities for amyloid formation.     

Recruitment of class I hydrophobins to the air:water interface initiates a multi-step process of functional amyloid formation

Class I fungal hydrophobins form amphipathic monolayers composed of amyloid rodlets. This is a remarkable case of functional amyloid formation in that a hydrophobic:hydrophilic interface is required to trigger the self-assembly of the proteins. The mechanism of rodlet formation and the role of the interface in this process have not been well understood. Here, we have studied the effect of a range of additives, including ionic liquids, alcohols, and detergents, on rodlet formation by two class I hydrophobins, EAS and DewA. Although the conformation of the hydrophobins in these different solutions is not altered, we observe that the rate of rodlet formation is slowed as the surface tension of the solution is decreased, regardless of the nature of the additive. These results suggest that interface properties are of critical importance for the recruitment, alignment, and structural rearrangement of the amphipathic hydrophobin monomers. This work gives insight into the forces that drive macromolecular assembly of this unique family of proteins and allows us to propose a three-stage model for the interface-driven formation of rodlets.     

Probing the Conformation of a Prion Protein Fibril with Hydrogen Exchange

A fragment of the prion protein, PrP(89–143, P101L), bearing a mutation implicated in familial prion disease, forms fibrils that have been shown to induce prion disease when injected intracerebrally into transgenic mice expressing full-length PrP containing the P101L mutation. In this study, we utilize amide hydrogen exchange measurements to probe the organization of the peptide in its fibrillar form. We determined the extent of hydrogen exchange first by tandem proteolysis, liquid chromatography, and mass spectrometry (HXMS) and then by exchange-quenched NMR. Although single amide resolution is afforded by NMR measurements, HXMS is well suited to the study of natural prions because it does not require labeling with NMR active isotopes. Thus, natural prions obtained from infected animals can be compared with model systems such as PrP(89–143, P101L) studied here. In our study, we find two segments of sequence that display a high level of protection from exchange, residues 102–109 and 117–136. In addition, there is a region that displays exchange behavior consistent with the presence of a conformationally heterogeneous turn. We discuss our data with respect to several structural models proposed for infectious PrP aggregates and highlight HXMS as one of the few techniques well suited to studying natural prions.     

Multiple substitutions of methionine 129 in human prion protein reveal its importance in the amyloid fibrillation pathway

The role of the polymorphism Met or Val in position 129 in the human prion protein is well documented regarding disease susceptibility and clinical manifestations. However, little is known about the molecular background to this phenomenon. We investigated herein the conformational stability, amyloid fibrillation kinetics, and seeding propensity of different 129 mutants, located in β-strand 1 of PrP (Met(129) (WT), M129A, M129V, M129L, M129W, M129P, M129E, M129K, and M129C) in HuPrP(90-231). The mutations M129V, M129L, M129K, and M129C did not affect stability (midpoints of thermal denaturation, T(m) = 65-66 °C), whereas the mutants M129A and M129E and the largest side chain M129W were destabilized by 3-4 °C. The most destabilizing substitution was M129P, which lowered the T(m) by 7.2 °C. All mutants, except for M129C, formed amyloid-like fibrils within hours during fibril formation under near physiological conditions. Fibril-forming mutants showed a sigmoidal kinetic profile and showed shorter lag times during seeding with preformed amyloid fibrils implicating a nucleated polymerization reaction. In the spontaneous reactions, the lag time of fibril formation was rather uniform for the mutants M129A, M129V, and M129L resembling the wild type. When the substituted amino acid had a distinct feature discriminating it from the wild type, such as size (M129W), charge (M129E, M129K), or rotational constraint (M129P), the fibrillation was impeded. M129C did not form ThT/Congo red-positive fibrils, and non-reducing SDS-PAGE of M129C during fibrillation conditions at different time points revealed covalent dimer formation already 15 min after fibrillation reaction initiation. Position 129 appears to be a key site for dictating PrP receptiveness toward recruitment into the amyloid state.     

Stabilization,Characterization, and Selective Removal of Cystatin C Amyloid Oligomers

The pathophysiological process in amyloid disorders usually involves the transformation of a functional monomeric protein via potentially toxic oligomers into amyloid fibrils. The structure and properties of the intermediary oligomers have been difficult to study due to their instability and dynamic equilibrium with smaller and larger species. In hereditary cystatin C amyloid angiopathy, a cystatin C variant is deposited in arterial walls and cause brain hemorrhage in young adults. In the present investigation, we use redox experiments of monomeric cystatin C, stabilized against domain swapping by an intramolecular disulfide bond, to generate stable oligomers (dimers, trimers, tetramers, decamers, and high molecular weight oligomers). These oligomers were characterized concerning size by gel filtration, polyacrylamide gel electrophoresis, and mass spectrometry, shape by electron and atomic force microscopy, and, function by assays of their capacity to inhibit proteases. The results showed the oligomers to be highly ordered, domain-swapped assemblies of cystatin C and that the oligomers could not build larger oligomers, or fibrils, without domain swapping. The stabilized oligomers were used to induce antibody formation in rabbits. After immunosorption, using immobilized monomeric cystatin C, and elution from columns with immobilized cystatin C oligomers, oligomer-specific antibodies were obtained. These could be used to selectively remove cystatin C dimers from biological fluids containing both dimers and monomers.     

Microcin amyloid fibrils A are reservoir of toxic oligomeric species

Microcin E492 (Mcc), a low molecular weight bacteriocin produced by Klebsiella pneumoniae RYC492, has been shown to exist in two forms: soluble forms that are believed to be toxic to the bacterial cell by forming pores and non-toxic fibrillar forms that share similar biochemical and biophysical properties with amyloids associated with several human diseases. Here we report that fibrils polymerized in vitro from soluble forms sequester toxic species that can be released upon changing environmental conditions such as pH, ionic strength, and upon dilution. Our results indicate that basic pH (≥8.5), low NaCl concentrations (≤50 mm), and dilution (>10-fold) destabilize Mcc fibrils into more soluble species that are found to be toxic to the target cells. Additionally, we also found a similar conversion of non-toxic fibrils into highly toxic oligomers using Mcc aggregates produced in vivo. Moreover, the soluble protein released from fibrils is able to rapidly polymerize into amyloid fibrils under fibril-forming conditions and to efficiently seed aggregation of monomeric Mcc. Our findings indicate that fibrillar forms of Mcc constitute a reservoir of toxic oligomeric species that is released into the medium upon changing the environmental conditions. These findings may have substantial implications to understand the dynamic process of interconversion between toxic and non-toxic aggregated species implicated in protein misfolding diseases.     

Relationship between prion propensity and the rates of individual molecular steps of fibril assembly

Peptides and proteins possess an inherent propensity to self-assemble into generic fibrillar nanostructures known as amyloid fibrils, some of which are involved in medical conditions such as Alzheimer disease. In certain cases, such structures can self-propagate in living systems as prions and transmit characteristic traits to the host organism. The mechanisms that allow certain amyloid species but not others to function as prions are not fully understood. Much progress in understanding the prion phenomenon has been achieved through the study of prions in yeast as this system has proved to be experimentally highly tractable; but quantitative understanding of the biophysics and kinetics of the assembly process has remained challenging. Here, we explore the assembly of two closely related homologues of the Ure2p protein from Saccharomyces cerevisiae and Saccharomyces paradoxus, and by using a combination of kinetic theory with solution and biosensor assays, we are able to compare the rates of the individual microscopic steps of prion fibril assembly. We find that for these proteins the fragmentation rate is encoded in the structure of the seed fibrils, whereas the elongation rate is principally determined by the nature of the soluble precursor protein. Our results further reveal that fibrils that elongate faster but fracture less frequently can lose their ability to propagate as prions. These findings illuminate the connections between the in vitro aggregation of proteins and the in vivo proliferation of prions, and provide a framework for the quantitative understanding of the parameters governing the behavior of amyloid fibrils in normal and aberrant biological pathways.     

Integrity of helix 2-helix 3 domain of the PrP protein is not mandatory for prion replication

The process of prion conversion is not yet well understood at the molecular level. The regions critical for the conformational change of PrP remain mostly debated and the extent of sequence change acceptable for prion conversion is poorly documented. To achieve progress on these issues, we applied a reverse genetic approach using the Rov cell system. This allowed us to test the susceptibility of a number of insertion mutants to conversion into prion in the absence of wild-type PrP molecules. We were able to propagate several prions with 8 to 16 extra amino acids, including a polyglycine stretch and His or FLAG tags, inserted in the middle of the protease-resistant fragment. These results demonstrate the possibility to increase the length of the loop between helices H2 and H3 up to 4-fold, without preventing prion replication. They also indicate that this loop probably remains unstructured in PrP(Sc). We also showed that bona fide prions can be produced following insertion of octapeptides in the two C-terminal turns of H2. These insertions do not interfere with the overall fold of the H2-H3 domain indicating that the highly conserved sequence of the terminal part of H2 is not critical for the conversion. Altogether these data showed that the amplitude of modifications acceptable for prion conversion in the core of the globular domain of PrP is much greater than one might have assumed. These observations should help to refine structural models of PrP(Sc) and elucidate the conformational changes underlying prions generation.     

Amyloid fibrils of human prion protein are spun and woven from morphologically disordered aggregates

Propagation and infectivity of prions in human prionopathies are likely associated with conversion of the mainly a-helical human prion protein, HuPrP, into an aggregated form with amyloid-like properties. Previous reports on efficient conversion of recombinant HuPrP have used mild to harsh denaturing conditions to generate amyloid fibrils in vitro. Herein we report on the in vitro conversion of four forms of truncated HuPrP (sequences 90–231 and 121–231 with and without an N-terminal hexa histidine tag) into amyloid-like fibrils within a few hours by using a protocol (phosphate buffered saline solutions at neutral pH with intense agitation) close to physiological conditions. The conversion process monitored by thioflavin T, ThT, revealed a three stage process with lag, growth and equilibrium phases. Seeding with preformed fibrils shortened the lag phase demonstrating the classic nucleated polymerization mechanism for the reaction. Interestingly, comparing thioflavin T kinetics with solubility and turbidity kinetics it was found that the protein initially formed non- thioflavionophilic, morphologically disordered aggregates that over time matured into amyloid fibrils. By transmission electron microscopy and by fluorescence microscopy of aggregates stained with luminescent conjugated polythiophenes (LCPs); we demonstrated that HuPrP undergoes a conformational conversion where spun and woven fibrils protruded from morphologically disordered aggregates. The initial aggregation functioned as a kinetic trap that decelerated nucleation into a fibrillation competent nucleus, but at the same time without aggregation there was no onset of amyloid fibril formation. The agitation, which was necessary for fibril formation to be induced, transiently exposes the protein to the air-water interface suggests a hitherto largely unexplored denaturing environment for prion conversion.Key words: misfolding, aggregation, amyloid, prion, conformational conversion, fluorescence