Quantifying collective interactions in biomolecular phase separation

Abstract

Biomolecular phase separation has emerged as a critical process for regulating cellular organisation and function. Modulators, ranging from ionic species to small molecule compounds and macromolecular binders, are key in controlling phase behaviour. The mechanisms of action of such modulators, however, have remained challenging to resolve as means to study protein-modulator interactions within condensates are lacking. Here we unravel biophysical mechanisms of protein phase separation modulation by applying a tie-line-gradient-based analysis approach to characterize the effects of modulators on the partitioning and interactions of species across the dense and dilute phases. Using the protein fused in sarcoma (FUS), we first characterize the impact of ionic species on FUS phase separation and find that potassium chloride (KCl) ions are preferentially excluded from condensates at low ionic strengths as they counteract intermolecular interactions by charge screening. Conversely, in the high salt reentrant phase separation regime, salts preferentially partition into the dense phase thereby scaffolding condensates by enhancing non-ionic interactions. Furthermore, we show that the common phase separation disruptor 1,6-hexanediol decreases FUS phase separation propensity by acting as a solvation agent for the protein. This leads to an expansion of the polypeptide chain, which limits the ability of FUS to form inter- and intramolecular interactions, thereby counteracting condensation. Lastly, we study a sequence-specific modulator of FUS phase separation using a FUS-targeting RNA aptamer and find a concentration-dependent response of condensate stabilisation already at nanomolar concentrations. The sequence-specific binder first partitions as a client molecule into the condensate and then acts as a scaffolder within the condensate. Taken together, our study highlights a broadly applicable approach for studying condensate partitioning allowing us to decipher the mechanisms of action of phase separation modulators.

Temperature-induced changes in protein interactions control RNA recruitment to G3BP1 condensates

“…The free energy difference of the phase separating system can be interpreted as a sum of contributions by each individual solute species. For each solute, the dominance factor D quantifies the relative weight of the contribution of the interactions of a solute to the whole system 45,46 (Fig. 2b).…”

“…LLPS is driven by the free energy difference between the homogeneous and the phase separated state. This energy difference underlies the differences in concentrations between the dilute and the dense phase which can be described by a tie-line vector 45,47 (Fig. 2a).…”

Expanding the molecular grammar of polar residues and arginine in FUS prion-like domain phase separation and aggregation

“…S4c) where the Csat for liquid droplets approaches that of the wild-type. These findings as a function of salt suggest the contribution of hydrophobic interactions to phase separation is enhanced at high salt with the more hydrophobic alanine-containing sequence 32,40,69 . Together, these results suggest that nontyrosine polar residue identity plays an important role in FUS phase separation and aggregation.…”

Biomolecular condensates sustain pH gradients at equilibrium driven by charge neutralisation

“…1c). 19,29,30 . Lower panels: Schematic representations visualising the proton partitioning as inferred from the reduced tie line gradients.…”

“…These unique microenvironments, often referred to as condensates, are believed to play critical roles in cellular organisation and pathology 14,16,17 . They can yield distinct material properties or differential molecular partitioning 18 , which has even been shown to extend to the formation of elemental ion gradients 19,20 . Reports even suggest that condensates could drive pH gradients intracellularly [21][22][23][24][25][26] .…”