Biology subjects

Kou, O. H.

Publications and source records attributed to Kou, O. H..

4 recordsLinked to original sources

bioRxiv2026

Cholesterol differentially regulates α-synuclein binding across membrane packing regimes

-Synuclein (Syn) 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 Syn 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 Syn binding preferences among reconstituted phosphatidylcholine/phosphatidylserine membranes with varied cholesterol content, lipid tail chemistry, and vesicle curvature. We found that cholesterols effect depended on the underlying packing regime of the membrane. In defect-rich membranes, cholesterol reduced Syn binding, consistent with cholesterol tightening lipid packing and reducing Syn-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 Syn binding. Instead, cholesterol regulates Syn-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 Syn binding. SignificanceSyn is a membrane-binding protein associated with Parkinsons disease, but the role of cholesterol in regulating its membrane interactions has remained unclear. Some studies report that cholesterol enhances Syn association with membranes, whereas others show that cholesterol suppresses it. This work helps explain why both outcomes can occur. We show that cholesterols effect depends on the membranes underlying packing state: cholesterol can reduce, enhance, or have little effect on Syn binding depending on the membrane environment. These findings shift the question from whether cholesterol is generally pro- or anti-binding to how cholesterol reshapes the membrane physical states that control Syn association.

biophysics↗

bioRxiv2026

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

-Synuclein (Syn) 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 Syn 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 Syn 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, Syn 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 Syn, 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 Syn-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.

biophysics↗

bioRxiv2026

The disordered and structured regions of α-Synuclein contribute to membrane remodeling synergistically

-Synuclein (Syn) remodels cellular membranes through interactions that involve both its structured, membrane-binding N-terminal domain (NTD) and intrinsically disordered C-terminal domain (CTD). While the amphipathic NTD helix is known to insert into lipid bilayers and generate curvature, the contribution of the acidic CTD remains unclear. Here, we dissect the individual and cooperative roles of these domains using Supported Bilayers with Excess Membrane Reservoir (SUPER) templates to quantify membrane remodeling via membrane fission and membrane morphological deformations (i.e., membrane budding and tubulation). We show that both the NTD and CTD independently remodel membranes, while full-length Syn exhibits greater remodeling ability than either the NTD or CTD in isolation. This result demonstrates a synergistic amplification between helix insertion of the NTD and the tethered, disordered CTD. To further probe the mechanism of membrane remodeling by the CTD, we modulated the chain length of the protein, the bulk ionic strength of the solution (i.e., charge screening), and applied relevant polymer scaling laws for disordered proteins. Our results suggest that the membrane remodeling mechanism for the disordered CTD is electrostatic in nature, stemming from protein-protein repulsion at elevated binding densities. Together, our findings reveal a cooperative energetic mechanism in which N-terminal helix insertion biases membrane curvature and the disordered, C-terminal domain adds an additional electrostatic component that helps to overcome the free energy barrier for membrane bending.

biophysics↗

bioRxiv2024

Lipid Packing Defects are Necessary and Sufficient for Membrane Binding of alpha-Synuclein

-Synuclein (Syn), an intrinsically disordered protein implicated in Parkinsons disease, is thought to initiate aggregation by binding to cellular membranes. Previous studies suggest that anionic lipids are necessary for this binding. However, these studies largely focused on unmodified Syn, while physiological Syn is N-terminally acetylated (NTA). Our work challenges the long-standing paradigm that anionic lipids are necessary for Syn binding by demonstrating that NTA diminishes Syns reliance on anionic membrane charge, revealing that membrane packing defects (i.e., interfacial hydrophobicity) alone can drive membrane binding. Using fluorescence microscopy and circular dichroism spectroscopy, we monitored the binding of NTA-Syn to membrane vesicles with different lipid compositions. Phosphatidylcholine and phosphatidylserine concentrations were varied to control surface charge, while phospholipid tail unsaturation and methylation were varied to modulate lipid packing. We also formulated cholesterol-containing membranes that mimicked the lipid composition of synaptic vesicles. In these membranes, all- atom molecular dynamics simulations were used to visualize and quantify membrane packing defects. Our results demonstrate that membrane packing defects are necessary for NTA-Syn binding and that defect-rich membranes are sufficient for NTA-Syn binding regardless of membrane charge. These findings provide a molecular mechanism by which lipid structural properties, such as poly-unsaturation, can regulate Syn binding to physiological membranes.

biophysics↗