Segmental ¹³ C-Labeling and Raman Microspectroscopy of α-Synuclein Amyloid Formation

Mapping conformational changes of α-synuclein (α-syn) from soluble, unstructured monomers to β-sheet- rich aggregates is crucial towards understanding amyloid formation. Raman microspectroscopy is now used to spatially resolve conformational heterogeneity of amyloid aggregates and monitor amyloid formation of segmentally ¹³ C-labeled α-syn in real-time. As the ¹³ C-isotope shifts the amide-I stretching frequency to lower energy, the ligated construct, ¹³ C_1-86¹² C_S87C-140 -α-syn, exhibits two distinct bands allowing for simultaneous detection of secondary structural changes in N-terminal 1-86 and C-terminal 87-140 residues. The disordered-to-β-sheet conformational change is first observed for the N-terminal followed by the C-terminal region. Finally, Raman spectroscopic changes occurred prior to Thioflavin T fluorescence enhancement, indicating that the amide-I band is a superior probe of amyloid formation.