Biology subjects

Johnson, D. H.

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

3 recordsLinked to original sources

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↗