(442j) Amphiphilic Proteins Coassemble into Multiphasic Condensates and Act As Biomolecular Surfactants

Cells contain membraneless compartments that assemble due to liquid–liquid phase separation, including biomolecular condensates with complex morphologies. We designed and examined families of amphiphilic proteins comprising one phase-separating domain and one non–phase- separating domain which paradoxically resemble biomembranes on membraneless organelles. In particular, these proteins contain the soluble structured domain glutathione S-transferase (GST) or maltose binding protein (MBP), fused to the intrinsically disordered RGG domain from P granule protein LAF-1. When one amphiphilic protein is mixed in vitro with RGG-RGG, the proteins assemble into enveloped condensates, with RGG-RGG at the core and the amphiphilic protein forming the surface film layer. In some configurations, these surfactant-like proteins influence condensate size. We also show that when both amphiphilic proteins are mixed together with the condensates, it results in MBP-based amphiphiles blocking adsorption of GST-based amphiphiles to the biomolecular condensates. Moreover, high concentration of GST-based ampihphiles results in phase separation of the proteins to form hierarchical structures. We also show that decoupling the domains of the amphiphilic protein with TEV protease dissolves the film layer. Our results suggest an important role of protein amphiphiles resembling biomembranes in establishing "membraneless" organelle structure and function.