Membrane Phase, Charge, and Curvature Regulate α-Synuclein Binding Dynamics.

α-Synuclein (αSyn) is an intrinsically disordered protein whose interactions with lipid membranes are central to both its physiological function and its role in synucleopathies. While membrane charge, phase, and curvature are each known to influence αSyn binding, these properties are typically examined independently, leaving their combined effects on both equilibrium and dynamic membrane association unresolved. Here, we systematically investigate how membrane phase and charge jointly regulate αSyn binding, curvature sensitivity, and exchange dynamics using fluorescence microscopy, circular dichroism spectroscopy, and fluorescence recovery after photobleaching (FRAP), complemented by coarse-grained molecular dynamics simulations. Under zwitterionic conditions, αSyn preferentially binds highly curved gel-phase membranes, driven by curvature-dependent enrichment of packing defects arising from faceted vesicle morphologies. Incorporation of anionic lipids selectively enhances binding in liquid-phase membranes while attenuating curvature-dependent partitioning in gel-phase membranes. Dynamic measurements reveal that membrane phase and charge also govern the stability of membrane-associated αSyn, with gel-phase membranes and anionic lipids promoting kinetically stabilized states. Simulations show that curvature-induced defect formation is strongly amplified in gel-phase membranes but largely insensitive to charge. These findings establish that αSyn-membrane interactions are governed by a cooperative interplay between membrane phase, curvature, and charge and highlight the importance of resolving both thermodynamic and kinetic contributions to protein-membrane binding.