Polyglutamine expansion within the exon1 of huntingtin leads to protein misfolding, aggregation, and cytotoxicity in Huntington's disease. This incurable neurodegenerative disease is the most prevalent member of a family of CAG repeat expansion disorders. Although mature exon1 fibrils are viable candidates for the toxic species, their molecular structure and how they form have remained poorly understood. Using advanced magic angle spinning solid-state NMR, we directly probe the structure of the rigid core that is at the heart of huntingtin exon1 fibrils and other polyglutamine aggregates, via measurements of long-range intramolecular and intermolecular contacts, backbone and side-chain torsion angles, relaxation measurements, and calculations of chemical shifts. These experiments reveal the presence of β-hairpin-containing β-sheets that are connected through interdigitating extended side chains. Despite dramatic differences in aggregation behavior, huntingtin exon1 fibrils and other polyglutamine-based aggregates contain identical β-strand-based cores. Prior structural models, derived from X-ray fiber diffraction and computational analyses, are shown to be inconsistent with the solid-state NMR results. Internally, the polyglutamine amyloid fibrils are coassembled from differently structured monomers, which we describe as a type of "intrinsic" polymorphism. A stochastic polyglutamine-specific aggregation mechanism is introduced to explain this phenomenon. We show that the aggregation of mutant huntingtin exon1 proceeds via an intramolecular collapse of the expanded polyglutamine domain and discuss the implications of this observation for our understanding of its misfolding and aggregation mechanisms.solid-state NMR | Huntington's disease | amyloid disease | protein aggregation | amyloid

Polyglutamine expansion in the huntingtin protein is the primary genetic cause of Huntington's disease (HD). Fragments coinciding with mutant huntingtin exon1 aggregate in vivo and induce HD-like pathology in mouse models. The resulting aggregates can have different structures that affect their biochemical behaviour and cytotoxic activity. Here we report our studies of the structure and functional characteristics of multiple mutant htt exon1 fibrils by complementary techniques, including infrared and solid-state NMR spectroscopies. Magic-angle-spinning NMR reveals that fibrillar exon1 has a partly mobile α-helix in its aggregation-accelerating N terminus, and semi-rigid polyproline II helices in the proline-rich flanking domain (PRD). The polyglutamine-proximal portions of these domains are immobilized and clustered, limiting access to aggregation-modulating antibodies. The polymorphic fibrils differ in their flanking domains rather than the polyglutamine amyloid structure. They are effective at seeding polyglutamine aggregation and exhibit cytotoxic effects when applied to neuronal cells.

Chiral nanoparticle assemblies are an interesting class of materials whose chiroptical properties make them attractive for a variety of applications. Here, C18-(PEPAuM-ox)2 (PEPAuM-ox = AYSSGAPPMoxPPF) is shown to direct the assembly of single-helical gold nanoparticle superstructures that exhibit exceptionally strong chiroptical activity at the plasmon frequency with absolute g-factor values up to 0.04. Transmission electron microscopy (TEM) and cryogenic electron tomography (cryo-ET) results indicate that the single helices have a periodic pitch of approximately 100 nm and consist of oblong gold nanoparticles. The morphology and assembled structure of C18-(PEPAuM-ox)2 are studied using TEM, atomic force microscopy (AFM), Fourier transform infrared (FTIR) spectroscopy, circular dichroism (CD) spectroscopy, X-ray diffraction (XRD), and solid-state nuclear magnetic resonance spectroscopy (ssNMR). TEM and AFM reveal that C18-(PEPAuM-ox)2 assembles into linear amyloid-like 1-D helical ribbons having structural parameters that correlate to those of the single-helical gold nanoparticle superstructures. FTIR, CD, XRD, and ssNMR indicate the presence of cross-β and polyproline II (PPII) secondary structure. A molecular assembly model is presented that takes into account all experimental observations and that supports the single-helical nanoparticle assembly architecture. This model provides the basis for the design of future nanoparticle assemblies having programmable structures and properties.

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Cataracts cause vision loss through the large-scale aggregation of eye lens proteins as a result of ageing or congenital mutations. The development of new treatments is hindered by uncertainty about the nature of the aggregates and their mechanism of formation. We describe the structure and morphology of aggregates formed by the P23T human γD-crystallin mutant associated with congenital cataracts. At physiological pH, the protein forms aggregates that look amorphous and disordered by electron microscopy, reminiscent of the reported formation of amorphous deposits by other crystallin mutants. Surprisingly, solid-state NMR reveals that these amorphous deposits have a high degree of structural homogeneity at the atomic level and that the aggregated protein retains a native-like conformation, with no evidence for large-scale misfolding. Non-physiological destabilizing conditions used in many in vitro aggregation studies are shown to yield qualitatively different, highly misfolded amyloid-like fibrils.