The conformational properties of soluble α‐synuclein, the primary protein found in patients with Parkinson's disease, are thought to play a key role in the structural transition to amyloid fibrils. In this work, we report that recombinant 100% N‐terminal acetylated α‐synuclein purified under mild physiological conditions presents as a primarily monomeric protein, and that the N‐terminal acetyl group affects the transient secondary structure and fibril assembly rates of the protein. Residue‐specific NMR chemical shift analysis indicates substantial increase in transient helical propensity in the first 9 N‐terminal residues, as well as smaller long‐range changes in residues 28–31, 43–46, and 50–66: regions in which the three familial mutations currently known to be causative of early onset disease are found. In addition, we show that the N‐terminal acetylated protein forms fibrils that are morphologically similar to those formed from nonacetylated α‐synuclein, but that their growth rates are slower. Our results highlight that N‐terminal acetylation does not form significant numbers of dimers, tetramers, or higher molecular weight species, but does alter the conformational distributions of monomeric α‐synuclein species in regions known to be important in metal binding, in association with membranes, and in regions known to affect fibril formation rates.

Significance: Cell-to-cell spreading of αS fibrils leads to amyloid seeding of endogenous monomer. Detailed atomic-level mechanistic understanding of the fibril seeding process of αS is essential for design of therapeutic approaches against Parkinson’s disease. In light of its complexity, this process remains ill defined at the molecular level. Using relaxation-based solution NMR experiments, we mapped a common N-terminal binding interface of the Ac-αS intrinsically disordered monomer with Ac-αS fibrils and off-pathway oligomers to elucidate critical monomer–aggregate interactions during seeded aggregation and in equilibrium with mature aggregates. From this work, we propose a paradigm in which Ac-αS monomer recruitment to the fibril is driven by interactions between the intrinsically disordered monomer N terminus and the flanking IDRs on the fibril surface.