Solution NMR of CsgC/CsgE interactions with ¹⁵N labeled α-synuclein.

<p>¹H-¹⁵N HSQC spectra at 10°C for 100 μM α-synuclein alone (red data in all panels) and upon addition of a 1-to-1 molar ratio of CsgC (<b>A</b>, blue) and CsgE (<b>B</b>, blue), and for a 1-to-5 molar ratio sample of CsgC and α-synuclein that had been shaken at 37°C for 48 h (<b>C,</b> blue). The data shown in <b>A</b> and <b>B</b> demonstrate that blue and red signals overlap except for His50 (<b>Insets</b> in <b>A</b> and <b>B</b>) and these spectra did not change over the course of three days. The visible chemical shifts in <b>C</b> were analyzed by NMR diffusion experiments to obtain an estimate of the molecular size. <b>D.</b> Analysis of perturbed residues in α-synuclein in the incubated CsgC-synuclein sample, based on the ¹H-¹⁵N HSQC peak intensities in <b>C</b> and reported assignments [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140194#pone.0140194.ref033" target="_blank">33</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140194#pone.0140194.ref034" target="_blank">34</a>]. Boxed residues represent chemical shifts assigned to specific residues (78 of the 140 residues were identified and used for the analysis). After shaking, 38 residues disappeared or broadened severely in the new species as judged from the peak intensities. Residues that broadened beyond detection are shown in red, and residues that lost > 90% of the original intensity are shown in yellow. Marked in bold are residues that show no apparent chemical shift change (Δω < 0.02 ppm, calculated as Δω = |0.2Δ¹⁵N+Δ¹H|).</p

Secondary structure of CsgC-induced oligomers.

<p>Far-UV CD spectra for 70 μM monomeric α-synuclein alone, and after shaking at 37°C for 48 h with and without 14 μM CsgC. The contribution from CsgC to the CD signal has been subtracted (CsgC shaken alone for 48 h did not result in any change of CD signal, data not shown). Whereas monomeric α-synuclein has random-coil like secondary structure, α-synuclein adopts β-sheet structure after incubation as a result of amyloid fiber formation. In the presence of CsgC, α-synuclein remains random-coil also after 48 h of shaking at 37°C.</p

Aggregation of α-synuclein in the presence of CsgC and CsgE.

<p><b>A.</b> ThT assay for α-synuclein aggregation with and without 1-to-10 molar ratio of CsgE:synuclein (red) or 1-to-10 molar ratio of CsgC:synuclein (blue). <b>B.</b> Bar graph showing the lag time for α-synuclein aggregation at 3 different ratios of CsgE and CsgC (1-to-3, 1-to-10, 1-to-100; 70 μM α-synuclein in all cases). ‘*’ Denotes no rise in ThT emission after 85 hrs. The error bars represent three experimental replicates. <b>C.</b> Fluorescence microscopy of end products of ThT assay for α-synuclein alone and for 1-to-3, 1-to-10, and 1-to-100 molar ratio of CsgE/CsgC-to-synuclein mixtures. Scale bar 100 μm. <b>D-F.</b> AFM images of end products after ThT experiments for α-synuclein alone (<b>D</b>), and in the presence of CsgE (<b>E</b>) and CsgC (<b>F</b>), as indicated. Scale bar 1 μm.</p