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 DAmFRET by creating a nanobody (mEosNb) against the fluorescent protein mEos3 to physically tether and thereby recruit candidate modifier proteins to mEos3-fused query proteins. This accessorization allows us to rapidly screen for effects on the phase behavior of query proteins by modulating the expression level and valency of mEosNb-fused modifiers. We show that our system recapitulates known effects of multivalency on liquid-liquid phase separation and can discriminate between nucleation mechanisms of amyloid and amyloid-like assemblies. Our approach adds a new experimental dimension for interrogating the mechanisms of intracellular phase transitions.