Formation of biomolecular condensates in bacteria by tuning protein electrostatics

Biomolecular condensates provide a strategy for cellular organization without a physical membrane barrier while allowing for dynamic, responsive organization of the cell. To date, very few biomolecular condensates have been identified in prokaryotes, presenting an obstacle to engineering these compartments in bacteria. As a novel strategy for bacterial compartmentalization, protein supercharging and complex coacervation were employed to engineer liquid-like condensates in E. coli. A simple model for the phase separation of supercharged proteins was developed and used to predict intracellular condensate formation. Herein, we demonstrate that GFP-dense condensates formed by expressing GFP variants of sufficient charge in cells are dynamic and enrich specific nucleic acid and protein components. This study provides a fundamental characterization of intracellular phase separation in E. coli driven by protein supercharging and highlights future utility in designing functional synthetic membraneless organelles.

Extremely supercharged proteins in mass spectrometry: profiling the pH of electrospray generated droplets, narrowing charge state distributions, and increasing ion fragmentation

High-performance solutions for supercharging proteins in electrospray ionization were optimized and the origin of the strong dependence of supercharging on acid strength was investigated.