Charge-Patterned Disordered Peptides Tune Intracellular Phase Separation in Bacteria

Abstract

Subcellular phase-separated compartments, known as biomolecular condensates, play an important role in the spatiotemporal organization of cells. To understand the sequence-determinants of phase separation in bacteria, we engineered protein-based condensates in Escherichia coli using electrostatic interactions as the main driving force. Minimal cationic disordered peptides were used to supercharge negative, neutral, and positive globular model proteins, enabling their phase separation with anionic biomacromolecules in the cell. The phase behavior was governed by the interaction strength between the cationic proteins and anionic biopolymers, in addition to the protein concentration. The interaction strength primarily depended on the overall net charge of the protein, but the distribution of charge between the globular and disordered domains also had an impact. Notably, the protein charge distribution between domains could tune mesoscale attributes such as the size, number, and subcellular localization of condensates within E. coli cells. The length and charge density of the disordered peptides had significant effects on protein expression levels, ultimately influencing the formation of condensates. Taken together, charge-patterned disordered peptides provide a platform for understanding the molecular grammar underlying phase separation in bacteria.

Selectivity of Complex Coacervation in Multi-Protein Mixtures

“…This suggests the potential of charged tags to tune or enhance complex coacervation based purification of proteins with low net charge globular domains. 43,44 Differences in the individual behavior of these GFPs suggests the importance of multivalent interactions as the isotropic distribution of charged residues is hypothesized to broaden the phase separation window due to the ability to make distinct interactions at multiple sites on the protein surface. However, in the mixture, GFP-tag, where half of the negative charge is localized to a single charge “patch” and therefore lacks additional interaction sites, behaved identically to GFP-11, indicating there were other factors at play in the mixture.…”