Figure 1.
Extracts of 20 ms DARR spectra highlight the sample quality.
All NMR spectra are from uniformly [13C,15N]-labeled α-synuclein fibrils and the electron micrographs are taken from the same batch of sample. (a) An early sample preparation resulting in a mixture of polymorphs. The spectrum was recorded at 14.1 T static magnetic field and 13 kHz MAS. (b) Spectra of preparations of pure polymorphs. Ribbons are shown in blue and fibrils in red. The spectra were recorded at 20.0 T static magnetic field and 17 kHz MAS. Full spectra are provided in ref. [20], [21]. All resonances of the fibrils and the ribbons plus additional resonances are present in the spectrum of the mixture.
Figure 2.
Ribbon and fibril feature entirely different spectra.
Overlay of the aliphatic region of a 20 ms DARR spectra of U [13C,15N] labeled α-synuclein ribbons (blue)[20] and α-synuclein fibrils (red)[21]. The spectra were recorded at 20.0 T static magnetic field and 17 kHz MAS and processed with a shifted sine-bell window function (SSB 2.6). The fibril spectrum is based on the same time-domain data as the DARR used for assignment. Individual spectra (with a slightly different processing) are given in the assignment notes [20], [21].
Figure 3.
Differences between secondary chemical shifts of Cα and Cβ resonances relative to their random-coil shifts as tabulated by Wishart and Sykes [27] of the sequentially assigned residues of α-synuclein. The data from the BMRB entries 17498 (ribbons) and 18860 (fibrils) were used for the plot. The secondary chemical shifts are shown in (a) blue for the ribbons and in (b) red and black for the fibrils, where black stands for the second of the doubled resonances starting at residue 79. The individual plots for the two polymorphs have already be presented in ref. [20], [21] and are replotted here to facilitate a direct comparison. The data for the ribbons differ slightly do to improved data obtained at 20 T. For glycine residues, only the deviation of the Cα shift from the random coil value is displayed. Glycines are indicated in light blue (ribbons) and in orange and grey (fibrils) for chain A and chain B, respectively. β-strands, as defined by three or more non-glycine residues in a row with a negative difference of the secondary shifts between Cα and Cβ are marked with arrows in dark blue or red, and lighter colors are used where glycine residues, which possibly are included into β-sheet, or where TALOS [63] predicted β-sheet, despite that the secondary chemical shift difference was positive. We only consider the dark stretches as trusted β sheets.
Figure 4.
Chains A and B signals have the same intensity.
Intensity ratio for selected doubled peaks using a 20 ms DARR spectra. Chain A is shown in black and chain B in grey. The errors are estimated from the experimental noise. All residues show a ratio close to 1:1 except for Gly86 and Tyr92 where the two forms may feature slightly different mobility.
Figure 5.
Ribbons and fibrils have an in-register parallel stacking.
(a) and (c) NCA (grey) and PAIN (red/blue) spectra of the fibril and the ribbon form, respectively. Peak maxima of the NCA spectra, taken from uniformly labeled samples, are labeled with grey crosses. Peaks that could unambiguously be assigned are labeled in the PAIN spectra obtained from 1:1 mixture of 13C and 15N labeled monomers. All spectra were recorded at 20.0 T static magnetic field and 17 kHz MAS. (b) and (d). Contacts visible in the PAIN spectrum are plotted against the sequence. ‘Clearly visible’ peaks could be assigned unambiguously in the PAIN and are labeled in (a) and (c), for ‘maybe visible’ ones there is intensity in the PAIN, however several assignments are possible, for ‘invisible’ ones no intensity could be observed at the expected positions. Regions supposedly in β-sheets regions according to TALOS and their secondary chemical shifts are highlighted in light red and light blue.
Figure 6.
The dynamics correlates with secondary-structure elements.
Intensity profile of CANCO cross peaks for (a) ribbons (blue) and (b) fibrils (red). The intensity reflects the local mobility, as all transfers used are CP steps and are therefore the intensity depends on T1ρ. Regions that are believed to be in β-sheets are highlighted.
Figure 7.
Residues 100 to 140 are highly dynamic.
1H-15N spectra of (a) ribbons (blue) and (b) fibrils (red). Black crosses mark the assigned resonances of the ribbon sample. The solution-state assignment of reference[42] can essentially be adopted but the assignment was verified with an HNCA assignment spectrum. All residues from Leu100 to the C-terminus, with exception of the prolines, are observed in the spectrum for the ribbons. For fibrils, these resonances are present as well. The black crosses in (b) mark the corresponding peak positions of the C-terminal resonances in the ribbon sample. Additional, weaker, signals are observed for fibrils, which likely come from α-synuclein monomers (see Figure S5 for details).
Figure 8.
Secondary structure of the two polymorphs described here compared to two other polymorphs of human α-synuclein [6], [24] and one form of mouse α-synuclein.
[16] The secondary structure for ribbons and fibrils is based on secondary chemical shifts and TALOS predictions. On top, the region that is usually assumed to form the fibrillar core is marked in blue. [31]