Biology subjects

Johnson, D. H.

Publications and source records attributed to Johnson, D. H..

6 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↗

bioRxiv2025

Recombinant protein platform for high-throughput investigation of peptide-liposome interactions via fluorescence anisotropy depolarization

Many cytosolic proteins critical to membrane trafficking and function contain an unstructured domain that can bind to specific membranes, with a transition into an amphipathic helix induced upon membrane association. These inducible amphipathic helices often play a critical role in organelle recognition and subsequent function by these cytosolic proteins, but the tools and techniques used to characterize affinity towards specific membranes are low-throughput and highly dependent on the solubility of the inducible amphipathic helix. Here, we introduce a modular recombinant protein platform for rapidly measuring the binding affinity of inducible amphipathic helices towards a variety of membrane compositions and curvatures using high-throughput fluorescence anisotropy measurements. Inducible amphipathic helices are solubilized with a fluorescently tagged small ubiquitin-like modifier (SUMO) protein and binding to membranes quantified by leveraging the unexpected decrease in fluorescence anisotropy upon binding, a phenomenon previously observed but not well understood. By using fluorescence anisotropy decay measurements and solution NMR experiments, we deduce that this phenomenon likely occurs due to the local increase in fluorophore motion upon binding to the membrane. Altogether, this recombinant protein platform can be readily applied to any inducible amphipathic helix of interest, allowing for detailed investigation of the specific membrane biochemical parameters facilitating binding.

molecular biology↗

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↗

bioRxiv2022

A comparison of survey method efficiency for estimating densities of Zebra Mussels (Dreissena polymorpha)

Abundance surveys are a common and practical technique to estimate plant or animal densities. Methods used to conduct these surveys often require jointly estimating uncertainty in both counts and detection probability. The estimation of detection probability requires additional measurements that take time, potentially reducing the efficiency of the survey for high-density populations. We conducted quadrat, removal, and distance surveys of zebra mussels (Dreissena polymorpha) in three central Minnesota lakes and used an efficiency measure to help compare survey designs. Survey efficiency depended on both the survey design and the population density. The efficiency of survey designs that required estimates of detection probabilities (relative to designs that did not) decreased with density, leading to a change in the most efficient survey design at high densities. These results demonstrate that the best survey design may be context-specific, requiring some prior knowledge of the underlying population density and the cost/time needed to collect additional information for estimating detection probabilities.

ecology↗