Using PICUP and targeted tyrosine mutations, the study maps transient intra- and inter-molecular contacts of α‑synuclein across solution, membrane-bound, oligomeric and fibrillar states, finding that membrane binding disrupts N–C intramolecular contacts while C‑terminal intermonomer contacts…

By defining state-specific α‑synuclein contact interfaces—especially persistent C‑terminal intermonomer interactions—this work clarifies mechanistic steps of aggregation, highlights a plausible target region for interventions or biomarker development, and provides a robust protocol useful for…

α-Synuclein (αSyn) is a neuronal protein predominantly found at the synapse, involved in vesicle trafficking. αSyn aggregates are also the main component of Lewy bodies, the hallmarks of Parkinson's disease. Interactions between the N- and C-termini of αSyn play crucial roles in its behavior in solution, membrane binding, and aggregation. Studying these interactions provides valuable insights into the physiological and pathological functions of αSyn. Here, we employed photoinduced cross-linking of unmodified proteins (PICUP) to identify the transient contacts of αSyn in different conformational states. By using tyrosine-to-phenylalanine mutations to block the reactivity of specific amino acids, we establish key cross-links in each state. In solution, we identify internal contacts between the N- and C-termini of monomers, as well as intermonomer contacts between C-termini in oligomers. When αSyn is bound to membranes, the internal cross-linking is blocked, while the cross-linking between C-terminal regions persists. In fibrils, cross-linking is significantly reduced, primarily occurring between the C-termini of adjacent monomers. This work highlights the effectiveness of PICUP for reporting on the transient contacts involved in αSyn self-assembly and its coassembly with lipid membranes, while providing a streamlined protocol that opens avenues for studying protein-protein interactions for a wide range of systems.