Abstract
Liquid-liquid phase separation (LLPS) of proteins that leads to formation of membrane-less organelles is critical to many biochemical processes in the cell. However, dysregulated LLPS can also facilitate aberrant phase transitions and lead to protein aggregation and disease. Accordingly, there is great interest in identifying small molecules that modulate LLPS. Here, we demonstrate that 4,4’-dianilino-1,1’-binaphthyl-5,5’-disulfonic acid (bis-ANS) and similar compounds are potent biphasic modulators of protein LLPS. Depending on context, bis-ANS can both induce LLPS de novo as well as prevent formation of homotypic liquid droplets. Our study also reveals the mechanisms by which bis-ANS and related compounds modulate LLPS and identify key chemical features of small molecules required for this activity. These findings may provide a foundation for the rational design of small molecule modulators of LLPS with therapeutic value.
Highlights
Liquid-liquid phase separation (LLPS) of proteins that leads to formation of membrane-less organelles is critical to many biochemical processes in the cell
Turbidity measurements used to assess the extent of LLPS and a phase diagram based on those measurements are shown in Supplementary Fig. 1a, b
These data clearly demonstrate that, at pH 6 in the absence of salt, the TDP-43 low complexity domain (LCD) remains in a single phase and that LLPS under these conditions is observed only above the saturation concentration of ~35 μM (Supplementary Fig. 1c, d)
Summary
Liquid-liquid phase separation (LLPS) of proteins that leads to formation of membrane-less organelles is critical to many biochemical processes in the cell. Our study reveals the mechanisms by which bis-ANS and related compounds modulate LLPS and identify key chemical features of small molecules required for this activity. 1234567890():,; Liquid−liquid phase separation (LLPS) mediates the formation of many intracellular membrane-less organelles (MLOs), including the nucleolus, nuclear paraspeckles, stress granules, and many other ribonucleoprotein granules[1,2,3] This diverse functionality over a large number of MLO subtypes reflects the capacity for LLPS to finely tune biochemical reactions through rapid assembly and disassembly of organelles as well as inclusion or exclusion of specific chemical components[1,4]. At higher RNA concentrations, decondensation (or a reentrant phase transition back into a single phase14,16) results from protein charge overscreening and, a long-range electrostatic repulsion[14] Such reentrant phase transitions are hypothesized to be a pervasive, general occurrence in many multivalent systems involving heterotypic interactions[18]. In contrast to classical complex coacervates in which the condensation and decondensation regimes reflect the number of charged residues within and the charge density of each polyelectrolyte, the decondensation regime for polypeptide-RNA systems is regulated by additional droplet-stabilizing, short-range cation−π interactions that become more significant at higher RNA to protein ratios[17,19,20]
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