Many diseases that degrade human health result from the aggregation of soluble unstructured proteins into insoluble amyloid fibrils, rich in β-sheets. For example, the aggregation of α-synuclein (α-syn), a 140-residue intrinsically disordered protein, into amyloid fibrils is implicated in the progression of Parkinson’s disease. Therefore, the ability to map conformational changes of α-syn from soluble, unstructured monomers to self-assembled, β-sheet-rich aggregates is crucial towards understanding amyloid pathogenicity. Here, we use Raman microspectroscopy, a vibrational spectroscopic technique, to spatially resolve conformational heterogeneity of amyloid aggregates and monitor amyloid formation of segmentally 13C-labeled α-syn in real-time. Native chemical ligation covalently links a uniformly 13C-labeled N-terminal portion containing residues 1–86 to its C-terminal 12C-complement residues 87–140. As the 13C-label shifts the carbonyl stretching frequency to lower energy, this ligated construct, 13C1-8612CS87C-140-α-syn, exhibits two distinct amide-I bands, which are highly sensitive to secondary structural changes. Using transmission electron microscopy, fibril morphologies for 13C1-8612CS87-140-α-syn are indistinguishable to those formed by uniformly 13C- and 12C-labeled-α-syn, indicating chemical ligation is minimally perturbative. Importantly, the disordered-to-β-sheet conformational change is first observed for the N-terminal followed by the C-terminal region. Unique Raman spectral evidence also revealed the presence of kinetic intermediates that contain β-sheet structure, at early times prior to the development of amyloid fibrils. Finally, through Raman mapping, we find the C-terminus structure is less uniform than the N-terminus in α-syn amyloid aggregates. By coupling segmental isotopic labeling and Raman spectroscopy, we have simultaneously obtained N- and C-terminal-specific conformational changes of α-syn during amyloid formation for the first time. Both molecular and mechanistic insights into α-syn amyloid formation are revealed and we anticipate that this strategy is broadly applicable to other studies of amyloid formation.

Segmental 13C-labeling and Raman microspectroscopy of α-synuclein amyloid formation

2019 Spring National Meeting

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