Structural Intermediates during α-Synuclein Fibrillogenesis on Phospholipid Vesicles

α-Synuclein (AS) fibrils are the main protein component of Lewy bodies, the pathological hallmark of Parkinson’s disease and other related disorders. AS forms helices that bind phospholipid membranes with high affinity, but no atomic level data for AS aggregation in the presence of lipids is yet available. Here, we present direct evidence of a conversion from α-helical conformation to β-sheet fibrils in the presence of anionic phospholipid vesicles and direct conversion to β-sheet fibrils in their absence. We have trapped intermediate states throughout the fibril formation pathways to examine the structural changes using solid-state NMR spectroscopy and electron microscopy. The comparison between mature AS fibrils formed in aqueous buffer and those derived in the presence of anionic phospholipids demonstrates no major changes in the overall fibril fold. However, a site-specific comparison of these fibrillar states demonstrates major perturbations in the <i>N</i>-terminal domain with a partial disruption of the long β-strand located in the 40s and small perturbations in residues located in the “non-β amyloid component” (NAC) domain. Combining all these results, we propose a model for AS fibrillogenesis in the presence of phospholipid vesicles

α‑Synuclein’s Adsorption, Conformation, and Orientation on Cationic Gold Nanoparticle Surfaces Seeds Global Conformation Change

α-Synuclein (α-syn), an aggregation-prone amyloid protein, has been suggested as a potential cause of Parkinson’s disease. When misfolded, α-syn aggregates as Lewy bodies in the brain, the loss of which can disrupt protein homeostasis. To investigate the potential of nanoparticle-mediated therapy for amyloid diseases, α-syn adsorption onto positively charged poly­(allylamine hydrochloride) coated gold nanoparticles (PAH Au NPs) was studied. α-Syn adsorbs in multilayers onto PAH Au NPs, which with increasing α-syn/PAH Au NP ratios (>2000 α-syn/PAH Au NP) results in the flocculation and sedimentation of α-syn coated PAH Au NPs. The orientation and conformation of α-syn on PAH Au NPs were studied using trypsin digestion and circular dichroism, which showed that α-syn adopts a random orientation on PAH Au NPs, with an increase in β-sheet and a decrease in α-helix structures. A consistent global change in α-syn’s conformation was also observed regardless of PAH Au NP concentration, suggesting bound α-syn initiates conformational changes to free α-syn

Study of Wild-Type α‑Synuclein Binding and Orientation on Gold Nanoparticles

The disruption of α-synuclein (α-syn) homeostasis in neurons is a potential cause of Parkinson’s disease, which is manifested pathologically by the appearance of α-syn aggregates, or Lewy bodies. Treatments for neurological diseases are extremely limited. To study the potential use of gold nanoparticles (Au NPs) to limit α-syn misfolding, the binding and orientation of α-syn on Au NPs were investigated. α-Syn was determined to interact with 20 and 90 nm Au NPs via multilayered adsorption: a strong electrostatic interaction between α-syn and Au NPs in the hard corona and a weaker noncovalent protein–protein interaction in the soft corona. Spectroscopic and light-scattering titrations led to the determinations of binding constants for the Au NP α-syn coronas: for the hard corona on 20 nm Au NPs, the equilibrium association constant was 2.9 ± 1.1 × 10⁹ M^–1 (for 360 ± 70 α-syn/NP), and on 90 nm Au NPs, the hard corona association constant was 9.5 ± 0.8 × 10¹⁰ M^–1 (for 5300 ± 700 α-syn/NP). The binding of the soft corona was thermodynamically unfavorable and kinetically driven and was in constant exchange with “free” α-syn in solution. A protease digestion method was used to deduce the α-syn orientation and structure on Au NPs, revealing that α-syn absorbs onto negatively charged Au NPs via its N-terminus while apparently retaining its natively unstructured conformation. These results suggest that Au NPs could be used to sequester and regulate α-syn homeostasis

Fibrils of mutant AS proteins prepared <i>in vitro</i> have a highly homogeneity and morphology similar to WT fibrils.

<p>(A) AS fibrils formation of (blue circles) E46K, (red triangles) A53T and (black squares) WT monitored by the Thioflavin T fluorescence assay. Error bars were determined from seven replicates for each. Measurements were normalized to the highest fluorescence intensity obtained across all samples. (B) Comparison of the electron micrographs of (top) E46K, (middle) A53T and (bottom) WT AS fibrils. ¹³C-¹³C 2D with 50 ms DARR mixing of (C) E46K and (D) A53T AS fibrils.</p

Sequential backbone-walk used to obtain the chemical shift assignments of A53T.

<p>Illustration of backbone connectivity through the NCACX (red), NCOCX (blue) and CAN(co)CX (black) spectra of residues A90-E83. In all cases the homonuclear mixing was achieved with 50 ms DARR.</p

TALOS+ predicted backbone dihedral angles ψ and φ as a function of residue number.

<p>(A) E46K and (B) A53T AS fibrils. Error bars based on the 10 best TALOS+ database matches. Representation of the secondary structure for WT (black), E46K (blue) and A53T (red) AS fibrils based on TALOS+ analysis (β-strands, arrows; turn or loop curved lines; not predicted, dashed lines). WT TALOS+ results based on those from Comellas <i>et al</i>.</p

The A53T mutation causes minor perturbations throughout the AS fibril sequence.

<p>(A) Expansions of ¹³C-¹³C 2D spectral overlays (50 ms DARR mixing, 600 MHz ¹H frequency and 13.3 kHz MAS) of WT (black) and A53T (red) AS fibril samples. (B) Plot of the chemical shift perturbations between WT and A53T chemical shifts versus residue number. Residues labeled as (*) correspond to perturbations above 1 ppm. Residues labeled as (#) correspond to glycines. The mutation is indicated with (†). Error bars correspond to the chemical shift variations from one WT batch to another. WT chemical shift assignments were obtained from the BMRB #16939.</p

The E46K mutation causes major chemical shift perturbations throughout the AS fibril sequence.

<p>(A) Expansions of ¹³C-¹³C 2D spectral overlays (50 ms DARR mixing) of WT (black) and E46K (blue) AS fibril samples. (B) Plot of chemical shift perturbations between WT and E46K chemical shifts versus residue number. Residues labeled as (*) correspond to perturbations greater than 5 ppm (¹⁵N) or 3 ppm (¹³C). Residues labeled as (#) correspond to glycines. The mutation is indicated with (†). Error bars correspond to the chemical shift variations from one WT batch to another. WT chemical shift assignments were obtained from the BMRB #16939.</p