Obermeyer, A. C. | bioRxiv Science

bioRxiv2021

Intracellular phase separation of globular proteins facilitated by short cationic peptides

Phase separation provides intracellular organization and underlies a variety of cellular processes. These biomolecular condensates exhibit distinct physical and material properties. Current strategies for engineering condensate formation include using intrinsically disordered domains and altering protein surface charge by chemical supercharging or site-specific mutagenesis. We add to this toolbox by designing short, highly charged peptide tags that provide several key advantages for engineering protein phase separation. Herein, we report the use of short cationic peptide tags for sequestration of proteins of interest into bacterial condensates. Using a panel of GFP variants, we demonstrate how cationic tag and globular domain charge contribute to intracellular phase separation in E. coli and observe that the tag can affect condensate disassembly at a given net charge near the phase separation boundary. We showcase the broad applicability of these tags by appending them onto enzymes and demonstrating that the sequestered enzymes remain catalytically active. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/450573v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1dc5380org.highwire.dtl.DTLVardef@1ee01a9org.highwire.dtl.DTLVardef@af0a85org.highwire.dtl.DTLVardef@1dba73d_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2020

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.

synthetic biology↗