Variations in prions, which cause different incubation times and deposition patterns of the prion protein isoform called PrP(Sc), are often referred to as 'strains'. We report here a highly sensitive, conformation-dependent immunoassay that discriminates PrP(Sc) molecules among eight different prion strains propagated in Syrian hamsters. This immunoassay quantifies PrP isoforms by simultaneously following antibody binding to the denatured and native forms of a protein. In a plot of the ratio of antibody binding to denatured/native PrP graphed as a function of the concentration of PrP(Sc), each strain occupies a unique position, indicative of a particular PrP(Sc) conformation. This conclusion is supported by a unique pattern of equilibrium unfolding of PrP(Sc) found with each strain. Our findings indicate that each of the eight prion strains has a PrP(Sc) molecule with a unique conformation and, in accordance with earlier results, indicate the biological properties of prion strains are 'enciphered' in the conformation of PrP(Sc) and that the variation in incubation times is related to the relative protease sensitivity of PrP(Sc) in each strain.

Studies using low-resolution fiber diffraction, electron microscopy, and atomic force microscopy on various amyloid fibrils indicate that the misfolded conformers must be modular, compact, and adopt a cross-␤ structure. In an earlier study, we used electron crystallography to delineate molecular models of the N-terminally truncated, disease-causing isoform (PrP Sc ) of the prion protein, designated PrP 27-30, which polymerizes into amyloid fibrils, but we were unable to choose between a trimeric or hexameric arrangement of right-or left-handed ␤-helical models. From a study of 119 all-␤ folds observed in globular proteins, we have now determined that, if PrP Sc follows a known protein fold, it adopts either a ␤-sandwich or parallel ␤-helical architecture. With increasing evidence arguing for a parallel ␤-sheet organization in amyloids, we contend that the sequence of PrP is compatible with a parallel left-handed ␤-helical fold. Left-handed ␤-helices readily form trimers, providing a natural template for a trimeric model of PrP Sc . This trimeric model accommodates the PrP sequence from residues 89 -175 in a ␤-helical conformation with the C terminus (residues 176 -227), retaining the disulfide-linked ␣-helical conformation observed in the normal cellular isoform. In addition, the proposed model matches the structural constraints of the PrP 27-30 crystals, positioning residues 141-176 and the N-linked sugars appropriately. Our parallel left-handed ␤-helical model provides a coherent framework that is consistent with many structural, biochemical, immunological, and propagation features of prions. Moreover, the parallel left-handed ␤-helical model for PrP Sc may provide important clues to the structure of filaments found in some other neurodegenerative diseases.

Results of transgenetic studies argue that the scrapie isoform of the prion protein (

Because the insolubility of the scrapie prion protein (PrP Sc ) has frustrated structural studies by x-ray crystallography or NMR spectroscopy, we used electron crystallography to characterize the structure of two infectious variants of the prion protein. Isomorphous two-dimensional crystals of the N-terminally truncated PrP Sc (PrP 27-30) and a miniprion (PrP Sc 106) were identified by negative stain electron microscopy. Image processing allowed the extraction of limited structural information to 7 Å resolution. By comparing projection maps of PrP 27-30 and PrP Sc 106, we visualized the 36-residue internal deletion of the miniprion and localized the N-linked sugars. The dimensions of the monomer and the locations of the deleted segment and sugars were used as constraints in the construction of models for PrP Sc . Only models featuring parallel ␤-helices as the key element could satisfy the constraints. These low-resolution projection maps and models have implications for understanding prion propagation and the pathogenesis of neurodegeneration.electron microscopy ͉ image processing ͉ Nanogold labeling ͉ parallel ␤-helix ͉ amyloid structure C reutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy (BSE), scrapie, and other spongiform encephalopathies are caused by an aberrantly folded isoform (PrP Sc ) of the prion protein (PrP) (1). Replication of prions includes a profound change in the conformation of the cellular isoform of PrP (PrP C ) to form the highly insoluble PrP Sc . The insolubility of PrP Sc has thwarted attempts to investigate its structure by either x-ray crystallography or NMR spectroscopy. Our knowledge about the structure of PrP Sc is therefore rather limited (2).After treatment with proteinase K (PK), PrP Sc loses the N-terminal residues 23 to Ϸ89 (forming PrP 27-30), but retains infectivity. During purification, PrP 27-30 polymerizes into rod-shaped filaments with the tinctorial properties of amyloid (3, 4). X-ray fibril diffraction illustrated the amyloid nature of PrP 27-30; characteristic 4.7 Å reflections indicative of cross-␤ structure were observed (5). Optical spectroscopy revealed that PrP Sc and PrP 27-30 are substantially enriched in ␤-sheet structure (6-9). This finding is in sharp contrast to the predominantly ␣-helical fold of the three-helix-bundle structure of PrP C as determined by NMR spectroscopy and x-ray crystallography on refolded recombinant PrP (10 -18). Owing to the lack of high-resolution structural information for PrP Sc , predictive methods have been used to develop molecular models to codify the existing spectroscopic, immunological, and biochemical data (19).In attempts to simplify the structural analysis of PrP Sc , we systematically deleted parts of the prion protein. One of these constructs containing only 106 residues, PrP106 (⌬23-88, ⌬141-176), supported the propagation of prions (20,21). Transgenic mice expressing only PrP106 develop a histologically accurate neurodegenerative prion disease after inoculation with prions, and the resulting prions can...

Recent evidence from several laboratories shows that the paired helical filaments of Alzheimer's disease brains consist mainly of the protein tau in an abnormally phosphorylated form, but the mode of assembly is not understood. Here we use EM to study several constructs derived from human brain tau and expressed in Escherichia coli. All constructs or tau isoforms are rodlike molecules with a high tendency to dimerize in an antiparallel fashion, as shown by antibody labeling and chemical crosslinking. The length of the rods is largely determined by the region of internal repeats that is also responsible for microtubule binding. One unit length of the repeat domain (three or four repeats) is around 22-25 nm, comparable to the cross-section of Alzheimer PHF cores. Constructs corresponding roughly to the repeat region of tau can form synthetic paired helical filaments resembling those from Alzheimer brain tissue. A similar self-assembly occurs with the chemically cross- linked dimers. In both cases there is no need for phosphorylation of the protein.

Many amyloid inhibitors resemble molecules that form chemical aggregates, which are known to inhibit many proteins. Eight known chemical aggregators inhibited amyloid formation of the yeast and mouse prion proteins Sup35 and recMoPrP in a manner characteristic of colloidal inhibition. Similarly, three known anti-amyloid molecules inhibited β-lactamase in a detergent-dependent manner, which suggests that they too form colloidal aggregates. The colloids localized to preformed fibers and prevented new fiber formation in electron micrographs. They also blocked infection of yeast cells with Sup35 prions, which suggests that colloidal inhibition may be relevant in more biological milieus.The aggregation of proteins into amyloid fibers is associated with a growing list of diseases, including diabetes, Alzheimer's, Parkinson's, Huntington's and the prion diseases. In these disorders, proteins aggregate into long, unbranched fibers after misfolding into a β-sheet-rich conformation 1 . Though there are no approved therapies targeting amyloid formation directly, many organic molecules inhibit fibrillization in vitro [2][3][4][5][6][7] . Some, such as the chelator clioquinol (1), even have activity in vivo 4 . These results have inspired the hope of therapeutic applications for some molecules 3-5 . Curiously, many fibrillization inhibitors resemble molecules known to form promiscuous chemical aggregates. These colloidal particles are composed of small organic molecules and range in size from 50 to over 600 nm 8 . Once formed, they physically sequester proteins and inhibit enzymes nonspecifically 8,9 . Like many inhibitors of amyloid polymerization, these colloidal inhibitors are typically highly conjugated, hydrophobic and dye-like (Supplementary Table 1 online) 8,9 . A good example is the amyloid inhibitor Congo red (2), a dye that was one of the first molecules observed to exhibit colloidal inhibition 8 . The flavonoid baicalein (3), an inhibitor of α-synuclein polymerization 6 , resembles the known chemical aggregator quercetin (4), and 4,5-dianilinophthalimide (DAPH, 5), an inhibitor of Alzheimer's amyloid formation 2 , resembles the aggregator bisindoylmaleimide (6 ; Supplementary Fig. 1 online).Given that chemical aggregates function through enzyme sequestration, we wondered whether they might also sequester protein molecules from each other, thereby preventing amyloid polymerization. Here, we investigate this hypothesis in two classic amyloid-forming proteins: the yeast prion protein Sup35 (ref. 10 ) and the recombinant mouse prion protein recMoPrP 89-230 (ref. 11 ). We ask whether known chemical aggregators can inhibit amyloid fiber formation, whether known fibrillization inhibitors form colloidal aggregates and whether amyloid inhibition by these molecules is in fact mediated via colloidal aggregation.Eight known chemical aggregators and two known nonaggregators 8,9 were tested for inhibition of Sup35 fibrillization in a thioflavin T (ThT, 7) fluorescence assay. All eight inhibited Sup35 fibrillization b...

A conformational isoform of the mammalian prion protein (PrP Sc ) is the sole component of the infectious pathogen that causes the prion diseases. We have obtained X-ray fiber diffraction patterns from infectious prions that show cross-␤ diffraction: meridional intensity at 4.8 Å resolution, indicating the presence of ␤ strands running approximately at right angles to the filament axis and characteristic of amyloid structure. Some of the patterns also indicated the presence of a repeating unit along the fiber axis, corresponding to four ␤-strands. We found that recombinant (rec) PrP amyloid differs substantially from highly infectious brainderived prions, both in structure as demonstrated by the diffraction data, and in heterogeneity as shown by electron microscopy. In addition to the strong 4.8 Å meridional reflection, the recPrP amyloid diffraction is characterized by strong equatorial intensity at approximately 10.5 Å, absent from brain-derived prions, and indicating the presence of stacked ␤-sheets. Synthetic prions recovered from transgenic mice inoculated with recPrP amyloid displayed structural characteristics and homogeneity similar to those of naturally occurring prions. The relationship between the structural differences and prion infectivity is uncertain, but might be explained by any of several hypotheses: only a minority of recPrP amyloid possesses a replication-competent conformation, the majority of recPrP amyloid has to undergo a conformational maturation to acquire replication competency, or inhibitory forms of recPrP amyloid interfere with replication during the initial transmission.amyloid ͉ protein ͉ neurodegeneration ͉ PrP ͉ ␤-helix

Prions are infectious proteins that encipher biological information within their conformations; variations in these conformations dictate different prion strains. Toward elucidating the molecular language of prion protein (PrP) conformations, we produced an array of recombinant PrP amyloids with varying conformational stabilities. In mice, the most stable amyloids produced the most stable prion strains that exhibited the longest incubation times, whereas more labile amyloids generated less stable strains and shorter incubation times. The direct relationship between stability and incubation time of prion strains suggests that labile prions are more fit, in that they accumulate more rapidly and thus kill the host faster. Although incubation times can be changed by altering the PrP expression level, PrP sequence, prion dose, or route of inoculation, we report here the ability to modify the incubation time predictably in mice by modulating the prion conformation.synthetic prions ͉ stability ͉ amyloid ͉ neurodegeneration ͉ conformation