Co-aggregation of α-synuclein with DOPC:DOPS 7∶3 (a, c) and DOPC (d, f) at L/P = 18 (molar ratio).

<p>Left: Lipid concentration derived from quantitative phosphorous analysis of aggregated and non-aggregated lipids after co-aggregation with 34 µM α-synuclein (a, d). Right: Polarization transfer solid state NMR on lamellar phase lipids (b, e) and lipids co-aggregated with α-synuclein (c, f). Stars indicate peaks originating from buffer molecules. Spectra are normalized to equal intensity for DP of C₁₈.</p

Water affects the molecular mobility of the SC components.

<p>¹³C MAS NMR spectra of intact SC as a function of RH (water content) at 32°C using DP (grey), CP (blue), and INEPT (red) pulse sequences for preferential enhancement of the signals from molecular segments in either rigid (CP) or mobile (INEPT) microenvironments. Prominent resonance lines from keratin (Leu C_β, Lys C_ε, Gly C_α, Ser C_α, Ser C_β) and lipids (all-trans and trans/gauche (CH₂)_n, (<i>ω</i>−1)CH₂, <i>ω</i>CH₃) are labeled in the data obtained at 50 wt% water. The dots in the 30 wt% water spectra indicate peaks of cholesterol (C12/24, C4, C14/17 and C9, cf., Fig. 2 <i>C</i>).</p

PT ssNMR spectra (DP black, CP blue, INEPT red) of DOPC:DOPS 7∶3 (top) and DOPC:DOPS 7∶3 co-aggregated with α-synuclein (bottom) at L/P = 18 (molar ratio).

<p>Numbers refer to assignments of the acyl chain from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077235#pone-0077235-g002" target="_blank">figure 2</a>. Spectra are scaled to equal DP intensities at 30–31 ppm.</p

Relative intensities from INEPT and CP experiments for DOPC:DOPS 7∶3 in lamellar phase (top) and co-aggregated with α-synuclein (lipid/protein ratio, L/P, 1/1; bottom) (DOPS is used in representation).

<p>Brackets indicate the unresolved group of peaks from C_4–7 together with C_12–15. I_INEPT/I_CP depends on both the correlation time τ_c and the order parameter |S_CH| for the bond vector in the molecular segment. Detailed interpretation is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077235#pone.0077235.s005" target="_blank">Table S1</a>.</p

Dynamic regimes and resulting signal intensities from PT ssNMR experiments.

<p>Theoretical ¹H to ¹³C polarization transfer efficiency as a function of correlation time <i>τ</i>_c and order parameter |<i>S</i>_CH| for a CH₂ segment at the magnetic field 11.74 T and the magic-angle spinning frequency 5 kHz, calculated with input parameters equal to the present experimental settings (see <i>Solid-state NMR</i>). The map is color-coded according to the calculated intensities of the INEPT (red) and CP (blue) polarization transfer schemes. White represents inefficient polarization transfer for both INEPT and CP. Typical values of <i>τ</i>_c and <i>S</i>_CH, in the different dynamic regimes, and the expected intensities for the INEPT and CP polarization transfer schemes, are listed to the right of the figure. Adopted from ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061889#pone.0061889-Nowacka2" target="_blank">[14]</a>.</p

Main acyl chain region of the PT ssNMR spectra (a–d) and cryo-TEM images (e–h), scale bars 200 nm) of α-synuclein fibrils co-aggregated with different amounts of DOPC:DOPS 7∶3 vesicles.

<p>Inserted boxed peaks are INEPT and CP signals for the unresolved peaks from C_4–7 and C_12–15 baseline adjusted for background protein CP signal. Spectra are scaled to give equal protein CP intensity at 20 ppm. Filled dark spots in the cryo-TEM images are frost defects and are not part of the experimental system. More representative images can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077235#pone.0077235.s003" target="_blank">Figure S3</a>.</p

Cryo-TEM images of α-synuclein fibrils formed after co-aggregation with different amounts of DOPC vesicles.

<p>Inserted boxed peaks are INEPT and CP signals for the unresolved peaks from C_4–7 and C_12–15 adjusted for protein CP signal by baseline correction. Spectra are scaled to give equal protein CP intensity at 20 ppm. Filled dark spots in cryo-TEM images are technique dependent frost defects and are not part of the experimental system. More representative images can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077235#pone.0077235.s004" target="_blank">Figure S4</a>.</p

Schematic representation of lipid-protein co-aggregation at different L/P ratios.

<p>α-synuclein alone aggregates to fibrils that form bundles. When aggregation takes place in the presence of lipid vesicles (L/P = 1), fibrillar co-aggregates composed of protein and lipids form. These aggregates arrange into mesh-like tangles. At higher L/P ratios, the excess lipid vesicles adsorb to the fibrils, and due to strong interaction with the surface, the vesicles are deformed to non-spherical shape.</p

Dynamic regimes and resulting intensities from polarisation transfer solid-state NMR experiments [30].

<p>Dynamic regimes and resulting intensities from polarisation transfer solid-state NMR experiments <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077235#pone.0077235-Nowacka2" target="_blank">[30]</a>.</p

Spearman rank correlation for serum VEGF-A concentration [pg/ml] before surgery and histological character of the tumor.

<p>Spearman rank correlation for serum VEGF-A concentration [pg/ml] before surgery and histological character of the tumor.</p