N-Terminal Amine Chemistry Influences α-Synuclein Interactions with Lipid Bilayers

High Resolution Image Download MS PowerPoint Slide α-Synuclein (αSyn) is an intrinsically disordered protein whose reversible membrane binding via amphipathic α-helix formation is central to its function and may also contribute to the pathology of Parkinson’s disease. In mammals, αSyn is constitutively N-terminally acetylated, a modification that alters its structural propensity and interactions with lipid bilayers. Here, we decouple the structural and chemical contributions of N-terminal acetylation by engineering several αSyn variants that systematically vary N-terminal helix propensity and physicochemical properties: unmodified αSyn (αSyn Unmod ), αSyn with an N-terminal Asn (αSyn Asn ), N-terminally acetylated αSyn (αSyn Acetyl ), and N-terminally succinylated αSyn (αSyn Succinyl ). Circular dichroism and NMR measurements show that all three modifications increase α-helical sampling within the first ∼10–12 residues, establishing a hierarchy of N-terminal helicity (αSyn Succinyl > αSyn Acetyl > αSyn Asn > αSyn Unmod ). However, fluorescence correlation spectroscopy reveals that enhanced helicity in solution does not strictly correlate with lipid bilayer affinity across several different lipid compositions. Instead, membrane binding is more strongly influenced by N-terminal chemistry, lipid composition, and bilayer curvature. Collectively, these results underscore how modifications tune αSyn’s interactions with binding partners, providing mechanistic insight into how this modification modulates αSyn function. For lipid bilayers, N-terminal acetylation tunes both the structural and physicochemical properties of αSyn to enhance binding.