A tool to dissect heterotypic determinants of homotypic protein phase behavior

Proteins commonly self‐assemble to create liquid or solid condensates with diverse biological activities. The mechanisms of assembly are determined by each protein's sequence and cellular context. We previously developed distributed amphifluoric FRET (DAmFRET) to analyze sequence determinants of self‐assembly in cells. Here, we extend the utility of DAmFRET by creating a nanobody (mEosNb) against the fluorescent protein mEos3 to physically tether other proteins to DAmFRET‐enabled query proteins. This tool allows us to rapidly screen for effects on the phase behavior of query proteins by modulating the expression level and valence of mEosNb‐fused modifier proteins. We use our system to identify thresholds of valence for liquid–liquid phase separation and to discriminate nucleation mechanisms of amyloid and other paracrystalline assemblies in cells. Our approach adds a new experimental dimension for interrogating the mechanisms of intracellular phase transitions.