Liquid Phase Separation Controlled by pH

Abstract

We present a minimal model to study the effects of pH on liquid phase separation of macromolecules. Our model describes a mixture composed of water and macromolecules that exist in three different charge states and have a tendency to phase separate. This phase separation is affected by pH via a set of chemical reactions describing protonation and deprotonation of macromolecules, as well as self-ionization of water. We consider the simple case in which interactions are captured by Flory-Huggins interaction parameters corresponding to Debye screening lengths shorter than a nanometer, which is relevant to proteins inside biological cells under physiological conditions. We identify the conjugate thermodynamic variables at chemical equilibrium and discuss the effective free energy at fixed pH. First, we study phase diagrams as a function of macromolecule concentration and temperature at the isoelectric point of the macromolecules. We find a rich variety of phase diagram topologies, including multiple critical points, triple points, and first-order transition points. Second, we change the pH relative to the isoelectric point of the macromolecules and study how phase diagrams depend on pH. We find that these phase diagrams as a function of pH strongly depend on whether oppositely charged macromolecules or neutral macromolecules have a stronger tendency to phase separate. One key finding is that we predict the existence of a reentrant behavior as a function of pH. In addition, our model predicts that the region of phase separation is typically broader at the isoelectric point. This model could account for both in vitro phase separation of proteins as a function of pH and protein phase separation in yeast cells for pH values close to the isoelectric point of many cytosolic proteins.

A generic approach to study the kinetics of liquid–liquid phase separation under near-native conditions

“…Next, we sought to generalize the pH-jump approach by demonstrating its rational extension to other proteins. First, we noted that the pH-regulated charge state of a protein is a general determinant of its LLPS 21 , as observed in many cases, such as lysozyme 22 , TDP-43 6 , Sup35 14 , and Pab1 8 . This effect can be rationalized by scrutinizing a typical phase diagram (Fig.…”

On the specificity of protein–protein interactions in the context of disorder

“…In all cases, the spatiotemporal confinement and the resulting population increase in the condensates, increase the concentration from typically low μM in the dilute phase, to high mM-mM concentrations in the dense phase [96][97][98][99][100]. Moreover, the condensates are dynamically formed and sensitive to environmental factors such as phosphorylation, pH and temperature, which can lead to their swift dissolution [101][102][103][104]. Thus, condensate formation may affect specificity in several ways.…”

Isolating and Analyzing Protein Containing Granules from Cells

“…1C). Studies have driven condensate formation using low temperatures, salt concentration, pH, and RNA concentration (Adame‐Arana, Weber, Zaburdaev, Prost, & Julicher, 2020; Kato et al., 2012; Lin, Protter, Rosen, & Parker, 2015; Riback et al., 2020; Sanders et al., 2020; Schwartz, Wang, Podell, & Cech, 2013). Molecular crowding can also be an important driver of phase separation and high‐molecular‐weight sugar polymers like polyethylene glycol and Ficoll can mimic the dense molecular environment within cells (Banani et al., 2017; Kato & McKnight, 2017; Molliex et al., 2015).…”