Structure and properties of alpha-synuclein and other amyloids determined at the amino acid level

Abstract

The structure of alpha-synuclein (alpha-syn) amyloid was studied by hydrogen-deuterium exchange by using a fragment separation-MS analysis. The conditions used made it possible to distinguish the exchange of unprotected and protected amide hydrogens and to define the order/disorder boundaries at close to amino acid resolution. The soluble alpha-syn monomer exchanges its amide hydrogens with water hydrogens at random coil rates, consistent with its natively unstructured condition. In assembled amyloid, long N-terminal and C-terminal segments remain unprotected (residues 1- approximately 38 and 102-140), although the N-terminal segment shows some heterogeneity. A continuous middle segment (residues approximately 39-101) is strongly protected by systematically H-bonded cross-beta structure. This segment is much too long to fit the amyloid ribbon width, but non-H-bonded amides expected for direction-changing loops are not apparent. These results and other known constraints specify that alpha-syn amyloid adopts a chain fold like that suggested before for amyloid-beta [Petkova et al. (2002) Proc. Natl. Acad Sci. USA 99, 16742-16747] but with a short, H-bonded interlamina turn. More generally, we suggest that the prevalence of accidental amyloid formation derives mainly from the exceptional ability of the main chain in a structurally relaxed beta-conformation to adapt to and energy-minimize side-chain mismatching. Seeding specificity, strain variability, and species barriers then arise because newly added parallel in-register chains must faithfully reproduce the same set of adaptations.

Fig. 1.

Characterization of α-syn. (A) Typical amyloid fibrils are seen by transmission electron microscopy (negative staining by uranyl acetate). (B)CD spectra for monomeric α-syn (random coil) and for suspensions of amyloid at pH 7 and a pDr of 4, the condition used for HX labeling experiments.

Fig. 2.

Illustration of the fragmentation–separation analysis (at pH 2.3 and 0°C). Samples of α-syn were fragmented by passage through two immobilized acid protease columns, and the fragments were roughly separated by reverse-phase HPLC and further resolved by continuous online scanning of the HPLC eluant by using electrospray Q-TOF MS. (A) MS-measured total ion current trace of the HPLC column eluant. (B) MS scan of the region marked in A. (C) Expansion of the mass peak marked in B. (D) The same peptide after deuterium labeling.

Fig. 3.

HX of α-syn (pDr of 4 and temperature of 5°C). D-labeling kinetics measured by the fragment separation method is shown for soluble α-syn monomer (green) and insoluble amyloid (red). Black curves compare the labeling expected for an unprotected random coil (10, 11). (Spreadsheet is available from the authors upon request.) Proline residues that have no exchangeable amide hydrogen are at positions 108, 117,120, 128, and 138. A low background of labeling through the amyloid region, in the range 1 ± 0.6 D per fragment, represents some unexplained experimental artifact. It is present from the earliest time point rather than accumulating at the free peptide rate, and its level is not correlated with fragment length.

Fig. 4.

HX protection map. Fragments measured are placed along the length of the α-syn molecule. Colors denote unprotected (green) and well protected (red) regions and fragments that overlap the boundaries (yellow).