CBE Doctoral Dissertation Defense: “Engineering Biomolecular Condensates: Insights Into Catalytic Activities and Structural Design” (Muyang Guan)

Abstract:

Eukaryotic cells orchestrate cellular processes through spatiotemporal organization achieved by compartmentalization. Biomolecular condensates, formed via liquid-liquid phase separation (LLPS), serve as vital hubs for enzymatic reactions and mediate cellular functions.

To investigate catalytic activities within biomolecular condensates, a light-emitting enzyme (NanoLuc) was incorporated into LAF-1 RGG model condensates. Incorporation into condensates led to a significantly enhanced enzymatic reaction rate for the two-phase system compared to the single-phase reaction. The reaction exhibited diffusion-limited behavior in the condensed phase, with light output affected by condensate viscosity. Given the low viscosity of our model condensates, this diffusion-limited behavior is anticipated in most condensates, suggesting the potential use of a condensate-NanoLuc system for identifying drug molecules that modulate condensate viscosity. In addition, complementation of enzyme fragments inside condensates and recruitment of cargo proteins at condensate surface was demonstrated.

To study the miscibility between different condensed phases, a second model condensate from the low complexity (LC) domain of human protein FUS was constructed. At physiological salt concentration, FUS LC and RGG condensates formed discrete condensed phases that are immiscible. The two condensates partially wet each other, indicating similar surface tensions. At high salt concentrations, the two phases formed a core-shell structure, suggesting the effect of charge interactions on interfacial tension. Additionally, viscoelasticity affected miscibility between two condensed phases. Further, dynamically driven substructures were observed as the system underwent out-of-equilibrium transition. This aspect can be harnessed to produce multiphasic condensates by kinetically trapping FUS LC inside RGG condensates. To systematically coordinate the interaction between the two phases, a designer “surfactant” protein was developed, reducing the FUS LC – RGG interfacial tension and enabling the integration of complex assemblies of two phases. These strategies demonstrated the ability to generate novel structures by bridging two phases that do not normally integrate, showcasing the potential for controlling condensate architecture.

In summary, this work provides valuable insights into the principles and strategies for modulating biomolecular condensate functions and structures, laying a foundation for further exploration and potential therapeutic applications.