Segmental ¹³C‐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 1–86 12 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.