RP-HPLC Chromatogram of the enzymatic evaluation of the model substrate.

<p>Conditions: lineal gradient of 0–100% MeCN (0.036% TFA) in H₂O (0.045% TFA) in 15 min. Flux of 1 mL/min, in a symmetry column C₁₈ (4.6 mm×150 mm), Waters, 100 Å, 5 µm, detection at 220 nm.</p

Representation of the peptide bound to PEGA₁₉₀₀ resin using the PLL linker (drawn in green).

<p>Representation of the peptide bound to PEGA₁₉₀₀ resin using the PLL linker (drawn in green).</p

General scheme of the peptides processed by the POP enzyme and the fragments generated.

<p>(a) Model substrate (b) <i>C</i>-terminal-anchored peptides (c) <i>N</i>-terminal-anchored peptides.</p

Representation of the linker system used in the (a) <i>C</i>-terminal analogs (b) <i>N</i>-terminal analogs.

<p>Representation of the linker system used in the (a) <i>C</i>-terminal analogs (b) <i>N</i>-terminal analogs.</p

Representation of the peptides used to confirm the esterase activity and capacity to process on solid-phase.

<p>(a) <i>C</i>-terminal-anchored analogs directly bounded on solid-phase. (b) <i>C</i>-terminal control peptides for the fluorescence assay directly bounded on solid-phase.</p

Representation of (a) the model substrate, (b) the <i>C</i>-terminal-anchored analogs and control peptides, (c) the <i>N</i>-terminal-anchored analogs and control peptides.

<p>Representation of (a) the model substrate, (b) the <i>C</i>-terminal-anchored analogs and control peptides, (c) the <i>N</i>-terminal-anchored analogs and control peptides.</p

RP-HPLC Chromatogram of an analog peptide (two spacers) before being anchored by the <i>N</i>-terminal side.

<p>Conditions: lineal gradient of 0–100% MeCN (0.036% TFA) in H₂O (0.045% TFA) during 15 min. Flux of 1 mL/min, in a symmetry column C₁₈ (4.6 mm×150 mm), Waters, 100 Å, 5 µm, detection at 220 nm.</p

Purities and HPLC elution times in a lineal gradient 0–100% MeCN-H₂0 (15 min), and MALDI-TOF MW of the synthesized peptides.

<p>Control cleavages are also reported for the peptides synthesized or reanchored into PEGA₁₉₀₀ resin.</p

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

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