Intracellular phase separation of globular proteins facilitated by short cationic peptides

Abstract

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 propose adding to this toolbox designer peptide tags that provide several potential advantages for engineering protein phase separation in bacteria. Herein, we demonstrate the use of short cationic peptide tags for sequestration of proteins of interest into bacterial condensates and provide a foundational study for their development as tools for condensate engineering. 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.

Charge-Patterned Disordered Peptides Tune Intracellular Phase Separation in Bacteria

“…Consistent with previous observations, the net charge was found to be a main governing parameter for condensate formation, and all variants with net charge ≥+12 showed observable condensates at 24 h postinduction. 32 Interestingly, GFP(−6)-L 2 also demonstrated intracellular phase separation at 24 h post-induction despite having a net charge of +6. The existence of a critical net charge for phase separation was also evident in in vitro turbidity assays using purified protein and total RNA from torula yeast (Figure S2F,G).…”

“…The use of minimal cationic peptides allowed us to preserve the relative and contextual importance of the globular domain in de novo condensates. We explored the interplay of contributions from both domains by systematically constructing a panel of proteins from three globular green fluorescent proteins (GFP) derived from superfolder GFP (sfGFP) with different surface charges, as has been done previously, 32 and, in this case, with two cationic disordered peptide motifs with varied charge-patterning. Specifically, we explored the charge, charge-patterning, and concentration dependencies of intracellular phase separation by overexpressing this panel of engineered proteins in E. coli to form de novo condensates.…”

Understanding the Impacts of Molecular and Macromolecular Crowding Agents on Protein–Polymer Complex Coacervates

“…75,76 The double-logarithmic plot of I(q) vs q was obtained for each sample and used for data analysis. The scattering vector q is defined as (7) where λ is the wavelength and 2θ is the scattering angle. For a system containing monodisperse, homogeneous, and isotropic dispersion of spherical particles, I(q) can be expressed as (8) where ϕ is the volume fraction of the particles, Δρ is the difference in scattering length density between the scattering particles and the solvent, V p is the volume of the particle, P(q) is the form factor providing information on the size and shape of the scattering object, and S(q) is the structure factor that is related to the spatial arrangements of particles and thus contains information on the interparticle interactions.…”

“…The resulting coacervate phase is enriched with interacting macromolecules and is in equilibrium with another liquid phase that is depleted in macromolecules. Complex coacervates can be prepared using a variety of macromolecules, including polysaccharides, − proteins, − nucleic acids, − and synthetic polymers. − Unique properties of complex coacervates, such as ultralow surface tension, high density, and tunable mechanical properties, − enable their use in a wide range of applications. Moreover, adjustment of the interactions between macromolecules can modify the properties of coacervates.…”

Glycation regulates phase separation by attenuating electrostatic interactions and increasing hydrophobic interactions

“…Recently, Obermeyer et al developed a strategy to engineer synthetic condensates using a short cationic peptide tag. 40 This cationic short peptide is rich in lysine and arginine, and it enables the linked proteins to phase separate by electrostatic interactions with the RNA in E. coli . Due to its suitable substrate and easily available intracellular system, this is a fit model for us to perform intracellular validation (Fig.…”