Thermodynamic and Structural Remodeling of Lipid Bilayers Induced by Gallic Acid

Neurodegenerative diseases and other pathological conditions are often associated with dysregulated protein phase separation, a process that leads to the formation of biomolecular condensates. These dynamic, membrane-free compartments play critical roles in cellular organization, but their abnormal transition into solid-like aggregates is a hallmark of several disorders. In this context, natural polyphenols, such as gallic acid (GA), have emerged as promising compounds capable of modulating the phase separation of intrinsically disordered proteins, such as tau and α-synuclein, inhibiting pathological aggregation. Furthermore, biomolecular condensates formed by specific proteins, like GRASP, present an opportunity to act as bio-inspired carriers for the targeted intracellular delivery of these pharmacological molecules. Understanding how GA interacts with cellular environments is a crucial first step for these applications. Here, we intend to investigate the biophysical interactions of GA with cell membrane models as a fundamental baseline before exploring its incorporation into GRASP condensates. We explored whether GA alters membrane organization, utilizing a comprehensive quantitative approach. We used differential scanning calorimetry (DSC), fluorescence spectroscopy, and electron paramagnetic resonance (EPR). To evaluate changes in membrane fluidity and hydration across thermal gradients, we reformulated the analysis of Laurdan fluorescence spectra using both the Generalized Polarization (GP) factor and spectral phasor approaches. Additionally, temperature-dependent EPR spectroscopy was performed using 5, 10, and 16-PC spin labels to detail the polarity and rotational mobility profiles at different depths of the lipid bilayer. Furthermore, computational simulations of the EPR spectra will be performed to extract further dynamic and structural parameters. Our quantitative results demonstrate that GA significantly modulates the thermodynamic behavior and structural dynamics of the membrane models. These findings provide critical biophysical parameters regarding GA's partitioning and stabilization at the lipid interface, establishing the necessary groundwork for our ongoing investigation into utilizing GRASP protein condensates as platforms for the encapsulation and release of therapeutic compounds.