Molecular-level secondary structure, polymorphism, and dynamics of full-length alpha-synuclein fibrils studied by solid-state NMR

Abstract

The 140-residue protein alpha-synuclein (AS) is able to form amyloid fibrils and as such is the main component of protein inclusions involved in Parkinson's disease. We have investigated the structure and dynamics of full-length AS fibrils by high-resolution solid-state NMR spectroscopy. Homonuclear and heteronuclear 2D and 3D spectra of fibrils grown from uniformly (13)C/(15)N-labeled AS and AS reverse-labeled for two of the most abundant amino acids, K and V, were analyzed. (13)C and (15)N signals exhibited linewidths of <0.7 ppm. Sequential assignments were obtained for 48 residues in the hydrophobic core region. We identified two different types of fibrils displaying chemical-shift differences of up to 13 ppm in the (15)N dimension and up to 5 ppm for backbone and side-chain (13)C chemical shifts. EM studies suggested that molecular structure is correlated with fibril morphology. Investigation of the secondary structure revealed that most amino acids of the core region belong to beta-strands with similar torsion angles in both conformations. Selection of regions with different mobility indicated the existence of monomers in the sample and allowed the identification of mobile segments of the protein within the fibril in the presence of monomeric protein. At least 35 C-terminal residues were mobile and lacked a defined secondary structure, whereas the N terminus was rigid starting from residue 22. Our findings agree well with the overall picture obtained with other methods and provide insight into the amyloid fibril structure and dynamics with residue-specific resolution.

Fig. 1.

CC correlation experiments using SD magnetization transfer, conducted on U-[¹³C,¹⁵N\K,V] AS fibrils. Measurements were performed at 18.8 T, at -8°C and a spinning speed of 12.5 kHz. Mixing-times for SD were 20 ms (red) and 150 ms (blue). Total experiment times were 5.5 h (red) and 8.5 h (blue); the maximum t₁ evolution times were 4 ms for both experiments. As an example, intraresidue (red) and interresidue (blue) cross-peaks for amino acids of the stretch G84–I88 are indicated. For a complete resonance assignment, see Table 1.

Fig. 2.

¹³C-MAS-NMR spectra recorded at 9.4 T at -5°C with a spinning speed of 8 kHz, with 512 scans each. Spectrum a, INEPT excitation; spectrum b, CP/MAS with a contact time of 1 ms; spectrum c, direct excitation with a 90° pulse on ¹³C; spectrum d, a transverse relaxation filter with a proton dephasing delay of 200 μs followed by a CP transfer of 1 ms.

Fig. 3.

Polymorphism in two different types of AS fibrils (red, batch A; green, batch B). (a) NCA correlation spectra. Spectra were recorded at 18.8 T, with a spinning frequency of 11 kHz at temperatures of -13°C. Maximum t₁ evolution times were 8 ms, and total experiment times were 4–5 h. (b and c) EM pictures of negatively stained fibrils of type A (b) and type B (c). The samples were stained with 1% uranyl acetate. (Scale bar: 400 Å; Inset: 200 Å.)

Fig. 4.

A 2D NHHC spectrum of AS fibrils (A form) grown from U-[¹³C,¹⁵N\K,V] AS diluted 1:2 with natural abundance AS. The spectrum was recorded at a static magnetic field of 18.8 T with a spinning speed of 11 kHz at a temperature of -13°C. The maximum t₁ evolution time was 4 ms, and the total experiment time was 113 h. The spectrum was processed with exponential multiplication with a line-broadening factor of 50 Hz in both dimensions. Red circles represent Nⁱ⁺¹C^αi cross-correlations for amino acids involved in β-strands according to chemical-shift analysis, including also Nⁱ⁺¹C^α(V)ⁱ cross-peaks due to residual carbon labeling in V residues. Red squares represent Nⁱ⁺¹C^αi cross-correlations for amino acids not involved in β-strands according to their chemical shift. Blue squares represent intraresidue N-C^α correlation for Gly residues. For several correlations, sequential residue numbers are given.

Fig. 5.

Characterization of the core region (residues 38–95) of AS. (a and b) φ (diamonds) and Ψ (squares) torsion angles classified as good by talos analysis for samples A (red) and B (green), respectively (with error bars as given). Filled symbols in a indicate angles, which were not classified as good by talos but were confirmed by Nⁱ⁺¹C^αi cross-correlations in the NHHC spectrum. White arrows indicate β-strands; gray lines are nonassigned amino acids; straight lines are assigned amino acids for which shift analysis did not give well-defined torsion angles; curved lines are turn or loop regions according to the NHHC spectrum; dashed lines indicate resonance doubling in sample B. (c) Absolute value of the ¹⁵N chemical shift differences between samples A and B. (d) rms deviation (rmsd) values for C^α and C^β shift differences between samples A and B, given as rmsd = |ΔδC^α| for Gly and [0.5(ΔδC^α2 + ΔδC^β2)]^1/2 for all other residues.