Amyloids are self-assembled protein architectures implicated in dozens of misfolding diseases. These assemblies appear to emerge through a "selection" of specific conformational "strains" which nucleate and propagate within cells to cause disease. The short Abeta(16-22) peptide, which includes the central core of the Alzheimer's disease Abeta peptide, generates an amyloid fiber which is morphologically indistinguishable from the full-length peptide fiber, but it can also form other morphologies under distinct conditions. Here we combine spectroscopic and microscopy analyses that reveal the subtle atomic-level differences that dictate assembly of two conformationally pure Abeta(16-22) assemblies, amyloid fibers and nanotubes, and define the minimal repeating unit for each assembly.

When the translation termination factor Sup35 adopts the prion state, [PSI + ], the read-through of stop codons increases, uncovering hidden genetic variation and giving rise to new, often beneficial, phenotypes. Evidence suggests that prion induction involves a process of maturation, but this has never been studied in detail. To do so, we used a visually tractable prion model consisting of the Sup35 prion domain fused to GFP (PrD-GFP) and overexpressed it to achieve induction in many cells simultaneously. PrD-GFP first assembled into Rings as previously described. Rings propagated for many generations before the protein transitioned into a Dot structure. Dots transmitted the [PSI + ] phenotype through mating and meiosis, but Rings did not. Surprisingly, the underlying amyloid conformation of PrD-GFP was identical in Rings and Dots. However, by electron microscopy, Rings consisted of very long uninterrupted bundles of fibers, whereas Dot fibers were highly fragmented. Both forms were deposited at the IPOD, a biologically ancient compartment for the deposition of irreversibly aggregated proteins that we propose is the site of de novo prion induction. We find that oxidatively damaged proteins are also localized there, helping to explain how proteotoxic stresses increase the rate of prion induction. Curing PrD-GFP prions, by inhibiting Hsp104's fragmentation activity, reversed the induction process: Dot cells produced Rings before PrD-GFP reverted to the soluble state. Thus, formation of the genetically transmissible prion state is a two-step process that involves an ancient system for the asymmetric inheritance of damaged proteins and heritable changes in the extent of prion fragmentation.yeast prion | fiber fragmentation | IPOD (Insoluble Protein Deposit) | prion inheritance | asymmetric damage distribution P rions are self-perpetuating protein conformations that store and transmit phenotypic information independently of nucleic acids. In fungi, they act as protein-based elements of heredity, stably propagating their altered protein conformations and associated phenotypes (1, 2).In Saccharomyces cerevisiae, seven prions are known (3) and evidence indicates that numerous other yeast proteins are capable of forming prions (4). The proteins have different molecular functions and produce different prion phenotypes. Although they share no sequence homology, their prion domains (PrDs) are enriched in asparagine and glutamine residues. These PrDs can adopt self-perpetuating prion conformations that are amyloids. They template the conversion of soluble prion proteins of the same type to the same conformation

Interest in the molecular structure of amyloid fibrils originates both from their association with many devastating diseases and as systems for exploring the energetics of higher order protein folding and assembly. These fibril arrays are generally viewed as rich in β-sheets, of either parallel or antiparallel orientation. [1][2][3][4][5] However, the relative arrangement of the sheets within the fibril remains poorly constrained in the existing structure models, 4,6,7 as these sheet-to-sheet arrangements are mediated predominantly by side chain packing. We now extend the use of metal ions as probes of amyloid side chain packing in simple segments of the Aβ peptide of Alzheimer's disease. By restricting the possible metal binding sites, we show that Zn 2+ can specifically control the rate of self-assembly and dramatically regulate amyloid morphology via distinct coordination environments.The histidine dyad, His13 and His14, of Aβ is implicated in metal binding 8,9 and the metalmediated toxicity of Aβ. [10][11][12][13] In a parallel, in-register β-sheet arrangement with sheet Hbonds oriented along the fibril axis (Figure 1a.), 3,4 the side chains of the His13 and His14 are spaced 5 Å apart along each surface of the β-sheets (Figure 1b). If the sheets are arrayed parallel to one another, the His13 and His14 side chains from different sheets are proximal, providing potential sites for Zn 2+ chelation along the sheets (Figure 1b), between the sheets (Figure 1c), or both. 6 Aβ(13-21), HHQKLVFFA, includes both the core segment, Aβ(17-21), known to be crucial for fibril formation, 14-18 and the metal binding dyad. To isolate His13/14 as the sole binding elements, the K16A peptide HHQALVFFA-NH 2 , Aβ(13-21)K16A, was prepared. As shown in Figure 2a, Aβ(13-21)K16A develops β-sheet secondary structure within 49 h, showing an increased mean residue molar ellipticity (MRME) by circular dichroism (CD) at 197 and 212 nm. The development of β-sheet structure was further confirmed by FTIR, showing the appearance of the amide I absorbance at 1628 cm −1 ( Figure S1a). TEM further established that Aβ(13-21)K16A assembles into fibrils (Fig. S1b), and these mature fibrils bind Congo red with the typical UV/vis absorption shift from 500 to 540 nm ( Figure S1c Figure 3). When the ribbons from the 1:1 incubation were pelleted, washed, and analyzed, the Zn 2+ to peptide ratio was 0.6-0.8 across three independent measurements. 19Wider 100-150 nm ribbons with variable twists form with longer incubation times (panels 2a, b, Figure 3). Some of the ribbons appear to coil and fuse to form tubular structures 200-300 nm in diameter (panels 2c, d, Figure 3). Therefore, Zn 2+ reduces the nucleation time of self-assembly across the entire concentration range and transforms Aβ(13-21)K16A assembly into either fibrillar or ribbon/ tubular morphology. 17 Struck by the different morphologies accessible to Aβ(13-21)-K16A, we investigated the coordination environment of Zn 2+ in the different assemblies by X-ray absorption spectroscopy (XAS). The soluble ...

Protein and peptide assembly into amyloid has been implicated in functions that range from beneficial epigenetic controls to pathological etiologies. However, the exact structures of the assemblies that regulate biological activity remain poorly defined. We have previously used Zn 2؉ to modulate the assembly kinetics and morphology of congeners of the amyloid ␤ peptide (A␤) associated with Alzheimer's disease. We now reveal a correlation among A␤-Cu 2؉ coordination, peptide self-assembly, and neuronal viability. By using the central segment of A␤, HHQKLVFFA or A␤(13-21), which contains residues H13 and H14 implicated in A␤-metal ion binding, we show that Cu 2؉ forms complexes with A␤(13-21) and its K16A mutant and that the complexes, which do not selfassemble into fibrils, have structures similar to those found for the human prion protein, PrP. N-terminal acetylation and H14A substitution, Ac-A␤(13-21)H14A, alters metal coordination, allowing Cu 2؉ to accelerate assembly into neurotoxic fibrils. These results establish that the N-terminal region of A␤ can access different metal-ion-coordination environments and that different complexes can lead to profound changes in A␤ self-assembly kinetics, morphology, and toxicity. Related metal-ion coordination may be critical to the etiology of other neurodegenerative diseases.copper-binding ͉ neurotoxicity ͉ self-assembly P rotein intermolecular assembly, especially formation of amyloid fibrillar structures, is correlated with a variety of human neurodegenerative diseases, including Alzheimer's, Parkinson's, Huntington's, and Creutzfeldt-Jakob diseases (1). More recently, amyloid has been tied to many nonpathological functional roles. For example, formation and self-perpetuation of amyloids in Saccharomyces cerevisiae regulate diverse yeast phenotypic expression as a positive response to environmental fluctuations (2), and amyloid may be involved in long-term memory and synapse maintenance in the marine snail, Aplysia (3, 4). Many proteins, including archetypical globular proteins such as myoglobin, can also form amyloid fibrils, suggesting that amyloidogenesis may be an intrinsic property of any ␣-amino acid polymer (5). Accordingly, these highly ordered paracrystalline protein self-assemblies have now been recognized as useful for nanostructure fabrication and biotechnology (6-8). Fully capturing these technological opportunities and understanding the biological roles of amyloid will depend on further definition of the organized structure and assembly pathway.Increasing evidence now implicates transition metal ions, including Zn 2ϩ , Cu 2ϩ , and Fe 3ϩ , as contributors both to amyloid ␤ (A␤) assembly in vitro and to the neuropathology of Alzheimer's disease, AD (9). The obligatory region of metal ion (Zn 2ϩ /Cu 2ϩ ) binding of A␤ has been mapped to the N terminus, amino acids 1-28 (10-16). In its soluble nonamyloid conformation, the peptide contains multiple intramolecular binding sites for Zn 2ϩ and Cu 2ϩ (9, 17), and intermolecular Zn 2ϩ binding can promote A␤ aggrega...

The role of Zn2+ in pre-organizing Abeta(10-21) amyloid formation is shown to preferentially alter the relative rate of fibril nucleation and to have little influence on fibril propagation. Fibril morphology, as determined by small angle neutron scattering (SANS) and transmission electron microscopy (TEM), was unchanged in the presence and absence of Zn2+ in Abeta(10-21), as well as in a series of site-specifically altered variants. The metal-independence of the Abeta(10-21)H13Q peptide suggested that the increase in nucleation rate in Abeta(10-21) is due to Zn2+-mediated inter-sheet interactions, involving both histidine 13 and histidine 14.

Amyloid fibrils are important in diverse cellular functions, feature in many human diseases and have potential applications in nanotechnology. Here we developed methods that combine optical trapping and fluorescent imaging to characterize the forces that govern the integrity of amyloid fibrils formed by a yeast prion protein. A critical advance was to employ the self-templating properties of amyloidogenic proteins to tether prion fibrils, enabling their manipulation in the optical trap. At normal pulling forces the fibrils were impervious to disruption. At much higher forces (up to 250 pN), discontinuities occurred in force-extension traces prior to fibril rupture. Selective amyloid disrupting agents and mutations demonstrated that such discontinuities resulted from the unfolding of individual subdomains. Thus, our results reveal unusually strong non-covalent intermolecular contacts that maintain fibril integrity, even when individual monomers partially unfold and extend fibril length.

No abstract

Prions are infectious, self-propagating protein aggregates that have been identified in evolutionarily divergent members of the eukaryotic domain of life. Nevertheless, it is not yet known whether prokaryotes can support the formation of prion aggregates. Here we demonstrate that the yeast prion protein Sup35 can access an infectious conformation in Escherichia coli cells and that formation of this material is greatly stimulated by the presence of a transplanted [PSI + ] inducibility factor, a distinct prion that is required for Sup35 to undergo spontaneous conversion to the prion form in yeast. Our results establish that the bacterial cytoplasm can support the formation of infectious prion aggregates, providing a heterologous system in which to study prion biology.Sup35 | [PSI + ] inducibility factor | amyloid P rions are infectious, self-propagating protein aggregates that have been implicated in a group of devastating mammalian neurodegenerative diseases (1). The discovery of a prion-like phenomenon in yeast and other fungi has led to profound advances in the understanding of prion biogenesis (2, 3). Prion proteins in mammals as well as fungi typically form highly structured β sheet-rich fibrils, known as amyloids, upon conversion to the infectious, prion form (4, 5, 1-3). However, unlike mammalian prions, yeast prions do not result in cell death, but instead act as heritable, protein-based genetic elements, conferring on the cell new phenotypic traits that are propagated epigenetically (6, 7). Although work over the last 15 years has uncovered a growing number of prions and prospective prion proteins in evolutionarily divergent members of the fungal kingdom (8-14), it is not yet known how pervasive prions are in nature; more specifically, it is not known whether bacteria contain prions or whether the bacterial cytoplasm can support the formation of prions. The study of yeast prions has revealed an essential interplay between prion proteins and cellular chaperone proteins (15, 16); thus, it is of particular interest to learn whether the bacterial chaperone environment is permissive for the formation of prion-like aggregates.To investigate whether the bacterial cytoplasm can support the formation of infectious amyloid, we sought to determine whether a yeast prion protein could access an infectious conformation in Escherichia coli cells. A particularly well-characterized prion in Saccharomyces cerevisiae, the [PSI + ] prion, is formed by the essential translation termination factor Sup35, which assembles into amyloid aggregates when it converts to the prion form (17; see also refs. 3 and 6). Upon conversion, the ability of Sup35 to participate in translation termination is impaired, and as a result, strains containing Sup35 in the prion form (designated [PSI + ] strains) manifest a nonsense-suppression phenotype due to significant stop-codon read-through (6). Like other yeast prion proteins, Sup35 has a modular structure, with a distinct priondetermining region (PrD) that is necessary to enable the protein to c...