(713c) Incorporation and Assembly of a Light-Emitting Enzymatic Reaction into Model Protein Condensates

Eukaryotic cells achieve specialized functions through subcellular compartmentalization, such as the sequestration and concentration of enzymes. Biomolecular condensates provide a strategy for compartmentalization through liquid-liquid phase separation (LLPS) of macromolecules such as proteins and nucleic acids, resulting the formation of a condensed phase isolated from the surrounding cellular environment. Emerging evidence suggests a role for LLPS and biomolecular condensates in human pathologies like neurodegenerative diseases, cancer, and viral replication. To study the enzymatic activities within biomolecular condensates, we utilized a model protein condensate system built from the RGG domain of the C. elegans P granule protein LAF-1. Herein, we demonstrate the incorporation of an ultrabright luciferase (NanoLuc) into model condensates that enables studies by bioluminescence microscopy and luminometry. We show that incorporation of NanoLuc significantly increased the overall reaction rate of the two-phase system compared to NanoLuc dispersion in the homogeneous phase. NanoLuc was estimated to be concentrated 10-fold inside these condensates. The resulting enzymatic reaction showed a diffusion-limited behavior, and the condensed phase behavior and light output was affected by condensate viscosity. As our model condensates have low viscosities, we expect the reaction to be diffusion-limited in most condensates and propose such a condensate-NanoLuc system for high-throughput screening (HTS) to identify drug molecules that alter the condensate viscosity. Further, we show that recruitment of complementary NanoLuc fragments into the condensates promoted their reconstitution into a functional full-length enzyme, indicating a liquid environment favoring protein-protein interactions (PPI). Lastly, we demonstrate cargo recruitment at the condensate-water interface through an engineered surfactant protein with coiled-coil interaction domain and achieve localized NanoLuc enzymatic activity therein, as well as the localization of other cargos. Overall, in this work, we demonstrate spatiotemporally regulated enzymatic activities through LLPS and protein self-assembly.