Residue-specific early-stage aggregation dynamics of α-synuclein (61-95) in monolayers: A pMAIRS and AFM study.

To better understand the misfolding behavior of α-synuclein (α-syn) at a controlled amphiphilic interface that models key features of membrane-associated confinement, we employed p-polarized Multiple-Angle Incidence Resolution Spectrometry (pMAIRS) in combination with Langmuir-Blodgett monolayer methods. The pMAIRS spectra were acquired at different time points during film compression to monitor the temporal evolution of supramolecular aggregation of the α-syn segment corresponding to residues 61-95 (α-syn(61-95)) at the air-water interface. Initially, α-syn(61-95) adopts an α-helical conformation with an overall tilt angle of approximately 31° to the interface. During two-hour compression, the peptide maintains its α-helical structure while undergoing gradual reorientation. The overall tilt angle decreases to 21.2°, indicating increased parallel alignment with the interface. Using 13C isotopic labeling at specific peptides residues, we observed that N-terminus residues (68G) transition from a perpendicular orientation to a more parallel configuration. In contrast, C-terminus residues (93G) remain consistently parallel to the interface throughout this process. Extended three-day compression reveals reporter-residue-specific conformational changes representing the N- and C-terminal regions of the NAC domain. N-terminus residues convert from α-helix to β-sheet conformation, as evidenced by the shift of the 13C amide I band from 1625 to 1616 cm-1 and its appearance exclusively in out-of-plane spectrum in pMAIRS. This indicates perpendicular orientation of backbone CO in these transformed residues. C-terminus residues (93G) retain their α-helical conformation throughout the extended incubation period. This creates conformationally asymmetric aggregates with distinct structural features. AFM imaging confirms the formation of early-stage protofibrils with structures that are morphologically and dimensionally distinct from mature fibers.