Cholesterol differentially regulates α-synuclein binding across membrane packing regimes.

α-Synuclein (αSyn) is an intrinsically disordered protein that preferentially binds anionic membranes with lipid packing defects. Cholesterol is an abundant membrane component that regulates packing and organization within membranes, yet its effect on αSyn binding remains unclear as prior studies report both cholesterol-mediated enhancement and suppression. Here, we investigated whether these conflicting effects reflect differences in the intrinsic packing state of the phospholipid bilayer. Using a quantitative fluorescence microscopy-based binding assay, we measured αSyn binding preferences among reconstituted phosphatidylcholine/phosphatidylserine membranes with varied cholesterol content, lipid tail chemistry, and vesicle curvature. We found that cholesterol's effect depended on the underlying packing regime of the membrane. In defect-rich membranes, cholesterol reduced αSyn binding, consistent with cholesterol tightening lipid packing and reducing αSyn-accessible defects. In membranes with intermediate defect content, cholesterol enhanced binding, whereas tightly packed membranes remained largely insensitive to cholesterol except when high cholesterol content was combined with high membrane curvature. Curvature further shaped these responses, with high curvature compressing cholesterol-dependent differences between membrane compositions. These results show that cholesterol does not universally promote or inhibit αSyn binding. Instead, cholesterol regulates αSyn-membrane interactions through a packing-regime-dependent mechanism shaped by both lipid tail chemistry and membrane curvature. This framework helps reconcile opposing reports in the literature and highlights membrane physical state as a key determinant of how cholesterol modulates αSyn binding.