Integrating experiments and simulations to unravel coacervate-membrane interactions: Insights into de-mixing and morphology modulation

Intrinsically disordered proteins and polypeptides can undergo liquid-liquid phase separation (LLPS) to form condensates/coacervates, which play numerous regulatory roles in the cell. Recently, the relevance of such LLPS occurring in the vicinity of membranes has been brought to light by several experimental studies. Membrane-adsorbed condensates are crucial for biomolecular localization, and in some cases, phase separation of proteins at the membrane surface induces significant changes in membrane morphology. A detailed microscopic understanding of the mechanisms behind these observations remains incomplete. Here we combine experiments and molecular simulations to unravel structural and dynamic features of the coacervate/membrane interface across scales. We study poly-Lysine/poly-Aspartate (K10/D10) coacervates as a prototype of phase-separated condensates with different unilamellar liposomes. Using a multiscale characterization approach that combines confocal microscopy, hyperspectral imaging, fluorescence recovery after photobleaching, and two complementary coarse-grained approaches, we show that the membrane-condensate affinity can be tuned by the anionic lipid content and quantified through the intrinsic contact angle - a material property derived from system geometry - both in vitro and in silico. We find that the membrane region in contact with the condensate displays a nearly two-fold reduced fluidity compared to the bare membrane. This is attributed to orientational ordering of lipid tails, resulting in decreased area per lipid. Moreover, we observed local lipid de-mixing induced by the coacervate adsorption. This study provides an effective framework for integrating experiment and computation to characterize the properties of coacervate/membrane interfaces that are critical to the functional impacts of these interactions.