Amphiphilic proteins coassemble into multiphasic condensates and act as biomolecular surfactants

Abstract

Significance Membraneless organelles are assemblies of highly concentrated biomolecules that condense through liquid–liquid phase separation. One major question in the field is how proteins assemble into multilayered condensates. Understanding mechanisms of formation of these systems is important for understanding the function and regulation of multiphasic organelles, such as P granules and nucleoli. A second outstanding question is how the size of biomolecular condensates is controlled. In this work, we generated amphiphilic proteins that localize to the surface of condensates. We observed diverse assemblies, including condensates enveloped by surfactant-like films, as well as complex multiphasic morphologies. In some configurations, these surfactant-like proteins influence condensate size. Our results suggest an important role of protein amphiphiles in establishing membraneless organelle structure and function. Cells contain membraneless compartments that assemble due to liquid–liquid phase separation, including biomolecular condensates with complex morphologies. For instance, certain condensates are surrounded by a film of distinct composition, such as Ape1 condensates coated by a layer of Atg19, required for selective autophagy in yeast. Other condensates are multiphasic, with nested liquid phases of distinct compositions and functions, such as in the case of ribosome biogenesis in the nucleolus. The size and structure of such condensates must be regulated for proper biological function. We leveraged a bioinspired approach to discover how amphiphilic, surfactant-like proteins may contribute to the structure and size regulation of biomolecular condensates. We designed and examined families of amphiphilic proteins comprising one phase-separating domain and one non–phase-separating domain. 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. Importantly, we found that MBP-based amphiphiles are surfactants and influence droplet size, with increasing surfactant concentration resulting in smaller droplet radii. In contrast, GST-based amphiphiles at increased concentrations coassemble with RGG-RGG into multiphasic structures. We propose a mechanism for these experimental observations, supported by molecular simulations of a minimalist model. We speculate that surfactant proteins may play a significant role in regulating the structure and function of biomolecular condensates.