Physics-Inspired Chemical Strategies for Probing and Modulating Biomolecular Condensates

Abstract

The physics of liquid-liquid phase separation (LLPS) underlies many biological functions of biomolecular condensates. However, the link between LLPS physics and condensate biology has been relatively understudied. For example, how physicochemical environment affects structure and conformation in condensate, which have distinct physicochemical properties, is unclear. Here, we show experimentally that network connectivity (percolation), a fundamental property of biomolecular condensates, can reshape conformational preferences in condensates. Inspired by Flory scaling, we developed a new method to estimate the conformational statistics of tracer polymers mimic protein’s intrinsically disordered regions (IDRs) by measuring how partition of the tracer scales with the length of the tracer. By replacing more structured RNA in condensates with less structured poly-A RNA, which confers increased percolation over time, we found that network connectivity mediated by RNA may cause more compact conformational statistics to become more favourable in condensates. By reconstituting the condensates under different salt concentrations, we found that network connectivity mediated by phase-separating protein may also cause a similar effect on conformational preferences. Thus, network connectivity, or percolation, emerges as an important variable for describing how structure and conformation depend on the environmental context in condensates (i.e. condensate structural context). As aberrant phase separation is emerging as a driver of cancers and degenerative diseases, the physics of LLPS also presents new possibilities for rational drug development targeting dysregulated biomolecular condensates. Observing that electrostatic interaction is a key physical consideration for LLPS, we developed a novel charge-driven strategy for directly modulating the phase separation behaviour of G3BP1 for inhibiting stress granule (SG) formation in cells. The G3BP1 study establishes a proof of concept for charge-based modulation of a phase-separating protein, which could expand the druggability of phase-separating proteins. Together, our work provides two proofs of concept for converting physical principles into chemically tractable measurements and perturbations, demonstrating both a new physics-biology relation and a novel strategy to leverage this relation for translational research.