Regulation of Elastin Liquid−Liquid Phase Separation with Increasing Ionic StrengthIdentification of Two Distinct Regimes

The formation of biomolecular condensates has emerged as a key feature of cellular organization. Despite their apparent ubiquity, the mechanisms underlying their regulation remain poorly understood. Biomolecular condensates form through liquid−liquid phase separation (LLPS) which predominantly involves intrinsically disordered proteins (IDPs). Here we show that the liquid−liquid phase separation boundary of α-elastin, an elastin-derived IDP, is sensitive to relatively small changes in the ionic strength and salt type. Across an extended ionic strength range, we identify two distinct regimes in which LLPS is first suppressed and then enhanced with increasing ionic strength. The lower ionic strength regime overlaps with the broad range encountered in biological solutions, whereas the higher ionic strength regime extends beyond physiological conditions. Using light scattering, we have characterized the net interprotein interactions, demonstrating that the remarkable sensitivity of the LLPS phase boundary to ionic strength is driven by the interplay between net weak protein−protein interactions and the thermodynamics of the solvent, which we explain using the framework of hydrophobic hydration. This work sheds light on the potential mechanisms by which cells regulate biomolecular condensation through subtle changes in the physicochemical properties of the solvent which can have a dramatic impact on protein phase separation propensity.