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The yeast prion protein Sup35 is a translation termination factor, whose activity is modulated by sequestration into a self-perpetuating amyloid. The prion-determining domain, NM, consists of two distinct regions: an amyloidogenic N terminus domain (N) and a charged solubilizing middle region (M). To gain insight into prion conversion, we used single-molecule fluorescence resonance energy transfer (SM-FRET) and fluorescence correlation spectroscopy to investigate the structure and dynamics of monomeric NM. Low protein concentrations in these experiments prevented the formation of obligate on-pathway oligomers, allowing us to study early folding intermediates in isolation from higher-order species. SM-FRET experiments on a dual-labeled amyloid core variant (N21C/S121C, retaining wild-type prion behavior) indicated that the N region of NM adopts a collapsed form similar to ''burst-phase'' intermediates formed during the folding of many globular proteins, even though it lacks a typical hydrophobic core. The mean distance between residues 21 and 121 was Ϸ43 Å. This increased with denaturant in a noncooperative fashion to Ϸ63 Å, suggesting a multitude of interconverting species rather than a small number of discrete monomeric conformers. (1), involving self-replicating or infectious protein conformations, has attracted broad interest in recent times due to its role in the biology of debilitating neurodegenerative diseases (1, 2), protein-based inheritance of novel phenotypes in yeast (3-5), and (potentially) long-term memory (6, 7). The Saccharomyces cerevisiae translational termination factor, Sup35, is one such protein capable of switching to a self-perpetuating state. In the prion state [PSI ϩ ], the glutamine/asparagine (Q/N)-rich prion domain of Sup35 is sequestered into an amyloid conformer, reducing the efficiencies of translation termination (8). This switch causes ribosomes to read through stop codons at biologically significant rates, changing a multitude of phenotypes (9).The NM segment (253 residues) of Sup35 determines the prion state and comprises two distinct regions. The N-terminal region (residues 1-123) is abundant in uncharged polar amino acids (glutamines, asparagines, and tyrosines), and forms the major part of the amyloid core that directs the protein into the [PSI ϩ ] prion state. The highly charged middle region M (residues 124-250) confers solubility in vitro and in vivo, allowing the protein to exist in the non-prion [psi Ϫ ] state. In the prion state, the N region adopts a ␤-sheet-rich conformation, whereas the M region remains relatively unstructured (10).Structural studies on NM amyloids have provided several insights into the molecular basis of prion nucleation (11)(12)(13)(14). An early step is the establishment of an equilibrium between predominantly unstructured NM polypeptide monomers and molten oligomeric intermediates that are obligate on-pathway species (14-16). The structure and dynamics of early monomeric intermediates are of considerable interest in deciphering the molecular m...

Formation of aberrant protein conformers is a common pathological denominator of different neurodegenerative disorders, such as Alzheimer's disease or prion diseases. Moreover, increasing evidence indicates that soluble oligomers are associated with early pathological alterations and that oligomeric assemblies of different disease-associated proteins may share common structural features. Previous studies revealed that toxic effects of the scrapie prion protein (PrP(Sc)), a β-sheet-rich isoform of the cellular PrP (PrP(C)), are dependent on neuronal expression of PrP(C). In this study, we demonstrate that PrP(C) has a more general effect in mediating neurotoxic signalling by sensitizing cells to toxic effects of various β-sheet-rich (β) conformers of completely different origins, formed by (i) heterologous PrP, (ii) amyloid β-peptide, (iii) yeast prion proteins or (iv) designed β-peptides. Toxic signalling via PrP(C) requires the intrinsically disordered N-terminal domain (N-PrP) and the GPI anchor of PrP. We found that the N-terminal domain is important for mediating the interaction of PrP(C) with β-conformers. Interestingly, a secreted version of N-PrP associated with β-conformers and antagonized their toxic signalling via PrP(C). Moreover, PrP(C)-mediated toxic signalling could be blocked by an NMDA receptor antagonist or an oligomer-specific antibody. Our study indicates that PrP(C) can mediate toxic signalling of various β-sheet-rich conformers independent of infectious prion propagation, suggesting a pathophysiological role of the prion protein beyond of prion diseases.

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SUMMARY Sequences rich in glutamine (Q) and asparagine (N) residues often fail to fold at the monomer level. This, coupled to their unusual hydrogen-bonding abilities, provides the driving force to switch between disordered monomers and amyloids. Such transitions govern processes as diverse as human protein-folding diseases, bacterial biofilm assembly, and the inheritance of yeast prions (protein-based genetic elements). A systematic survey of prion-forming domains suggested that Q and N residues have distinct effects on amyloid formation. Here we use cell biological, biochemical, and computational techniques to compare Q/N-rich protein variants, replacing Ns with Qs and Qs with Ns. We find that the two residues have strong and opposing effects: N-richness promotes assembly of benign self-templating amyloids; Q-richness promotes formation of toxic non-amyloid conformers. Molecular simulations focusing on intrinsic folding differences between Qs and Ns suggest that their different behaviors are due to the enhanced turn-forming propensity of Ns over Qs.