Recombinant mouse prion protein (recMoPrP) produced in Escherichia coli was polymerized into amyloid fibrils that represent a subset of β sheet–rich structures. Fibrils consisting of recMoPrP(89–230) were inoculated intracerebrally into transgenic (Tg) mice expressing MoPrP(89–231). The mice developed neurologic dysfunction between 380 and 660 days after inoculation. Brain extracts showed protease-resistant PrP by Western blotting; these extracts transmitted disease to wild-type FVB mice and Tg mice overexpressing PrP, with incubation times of 150 and 90 days, respectively. Neuropathological findings suggest that a novel prion strain was created. Our results provide compelling evidence that prions are infectious proteins.

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In vivo under pathological conditions, the normal cellular form of the prion protein, PrP C (residues 23-231), misfolds to the pathogenic isoform PrP Sc , a ␤-rich aggregated pathogenic multimer. Proteinase K digestion of PrP Sc leads to a proteolytically resistant core, PrP 27-30 (residues 90 -231), that can form amyloid fibrils. To study the kinetic pathways of amyloid formation in vitro, we used unglycosylated recombinant PrP corresponding to the proteinase K-resistant core of PrP Sc and found that it can adopt two non-native abnormal isoforms, a ␤-oligomer and an amyloid fibril. Several lines of kinetic data suggest that the ␤-oligomer is not on the pathway to amyloid formation. The preferences for forming either a ␤-oligomer or amyloid can be dictated by experimental conditions, with acidic pH similar to that seen in endocytic vesicles favoring the ␤-oligomer and neutral pH favoring amyloid. Although both abnormal isoforms have high ␤-sheet content and bind 1-anilinonaphthalene-8-sulfonate, they are dissimilar structurally. Multiple pathways of misfolding and the formation of distinct ␤-sheet-rich abnormal isoforms may explain the difficulties in refolding PrP Sc in vitro, the need for a PrP Sc template, and the significant variation in disease presentation and neuropathology.

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Prion disease is a neurodegenerative malady, which is believed to be transmitted via a prion protein in its abnormal conformation (PrPSc). Previous studies have failed to demonstrate that prion disease could be induced in wild-type animals using recombinant prion protein (rPrP) produced in Escherichia coli. Here, we report that prion infectivity was generated in Syrian hamsters after inoculating full-length rPrP that had been converted into the cross-β-sheet amyloid form and subjected to annealing. Serial transmission gave rise to a disease phenotype with highly unique clinical and neuropathological features. Among them were the deposition of large PrPSc plaques in subpial and subependymal areas in brain and spinal cord, very minor lesioning of the hippocampus and cerebellum, and a very slow progression of disease after onset of clinical signs despite the accumulation of large amounts of PrPSc in the brain. The length of the clinical duration is more typical of human and large animal prion diseases, than those of rodents. Our studies establish that transmissible prion disease can be induced in wild-type animals by inoculation of rPrP and introduce a valuable new model of prion diseases.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-009-0633-x) contains supplementary material, which is available to authorized users.

A growing number of biologically important proteins have been identified as fully unfolded or partially disordered. Thus, an intriguing question is whether such proteins can be forced to fold by adding solutes found in the cells of some organisms. Nature has not ignored the powerful effect that the solution can have on protein stability and has developed the strategy of using specific solutes (called organic osmolytes) to maintain the structure and function cellular proteins in organisms exposed to denaturing environmental stresses (Yancey, P. H., Clark, M. E., Hand, S. C., Bowlus, R. D., and Somero, G. N. (1982) Science 217, 1214 -1222). Here, we illustrate the extraordinary capability of one such osmolyte, trimethylamine N-oxide (TMAO), to force two thermodynamically unfolded proteins to fold to nativelike species having significant functional activity. In one of these examples, TMAO is shown to increase the population of native state relative to the denatured ensemble by nearly five orders of magnitude. The ability of TMAO to force thermodynamically unstable proteins to fold presents an opportunity for structure determination and functional studies of an important emerging class of proteins that have little or no structure without the presence of TMAO.A growing number of biologically important proteins have been identified as fully or partially disordered under physiological conditions (e.g. different classes of DNA-binding proteins (1), transactivation domains of transcription factors (2-6), non-A␤ component of Alzheimer's disease amyloid plaque precursor implicated in Alzheimer's disease (7), and others (8,9). The issue of shifting a protein or domain from an unfolded to a folded ensemble is a topic of interest not only for these proteins, but also for a host of marginally stable proteins. A question of interest is whether such proteins can be induced to adopt unique and functionally important ordered structures by addition of solutes found in the cells of some organisms.According to Anfinsen, "The native conformation of protein is determined by the totality of interatomic interactions and by the amino acid sequence, in a given environment " (10). Although the statement by Anfinsen acknowledges the importance of both amino acid sequence and the physiological milieu in defining the native (Gibbs energy minimum) conformation of proteins, the overwhelming emphasis in the protein folding field has been on the interatomic interaction aspect of the process (11). Nature, however, has not ignored the powerful effect that the solution can have on protein stability and has developed the strategy of using specific solutes (called organic osmolytes) to maintain the structure of proteins in cells exposed to denaturing environmental stresses (12). Thus, through the power of natural selection, solutes were evolved that have exceptional ability to promote the native states of proteins in the presence of denaturing stresses. The implication is that in the absence of denaturing stresses, osmolytes continue to exert a force t...

The recombinant mouse prion protein (MoPrP) can be folded either to a monomeric ␣-helical or oligomeric ␤-sheet-rich isoform. By using circular dichroism spectroscopy and size-exclusion chromatography, we show that the ␤-rich isoform of MoPrP is thermodynamically more stable than the native ␣-helical isoform. The conformational transition from the ␣-helical to ␤-rich isoform is separated by a large energetic barrier that is associated with unfolding and with a higher order kinetic process related to oligomerization. Under partially denaturing acidic conditions, MoPrP avoids the kinetic trap posed by the ␣-helical isoform and folds directly to the thermodynamically more stable ␤-rich isoform. Our data demonstrate that the folding of the prion protein to its native ␣-helical monomeric conformation is under kinetic control.Although protein folding is commonly thought to be controlled by thermodynamic preferences, it has been understood by many, including Anfinsen and others (1,2), that kinetic issues can alter the folding landscape. Whereas most small globular proteins will refold spontaneously in vitro to a native conformation, in vivo folding often exploits auxiliary molecules and defined subcellular compartments to avoid the deposit of misfolded forms (3). Increasingly, a role for protein misfolding in a variety of neurodegenerative diseases has emerged. A common thread joining prion-based diseases and Alzheimer's disease, and possibly Parkinson's disease and frontotemporal dementia, is the conversion of a normal, cellular, monomeric isoform of a protein into a ␤-sheet-rich, polymeric form (4 -6). When the deposited polymeric form is sufficiently ordered to bind Congo red and exhibit birefringence to polarized light, the pathologic term amyloid is used to cluster these and other maladies (7).Recent studies by Dobson and others (8 -12) have demonstrated that a broad variety of proteins that rapidly fold into monomeric or oligomeric cellular forms under native-like conditions can also be refolded into ␤-rich, amyloid forms under conditions that destabilize the native state. So far, these proteins have not been associated with human deposition diseases. This finding has led to the suggestion that the ability to adopt alternative ␤-rich folds capable of forming amyloid is not a unique property of specific proteins associated with conformational diseases but reflects a general property of polypeptide chains (13). The interplay between protein concentration and the conformational preferences of the monomeric chain in driving the transition to a ␤-rich multimeric isoform remains to be more fully explored.Glockshuber and colleagues (14) have shown that a fragment of the mouse prion protein folds very rapidly into the ␣-helixrich conformation with a half-life of 170 s as measured at 4°C. Here, we report that a ␤-sheet-rich conformation of the mouse prion protein (MoPrP) 1 is thermodynamically more stable than its native ␣-helix-rich conformation. The conformational transition from the ␣-helical to a ␤-sheet-rich isofor...

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