(636h) Redox Sensitive Protein Droplets from Recombinant Oleosin

Molecular biology enables the creation of novel biomaterials with designer functionality and tailored responsiveness. In this work, we designed a protein that at low concentrations forms nanometer sized spherical micelles, and at high concentrations forms micron sized liquid droplets. Our work is based on the plant surfactant protein, oleosin. Oleosin, akin to a triblock copolymer, contains N and C-terminal hydrophilic regions and a central hydrophobic region. At low concentrations, the hydrophobic regions drives the formation of spherical micelles. Whereas, at higher protein concentrations, oleosin condenses into liquid droplets below a critical temperature. These liquid droplets are dynamic structures that fuse upon contact with one another and whose phase behavior is thermo-reversible, similar to many naturally occurring membrane-less organelles. In this work we explore strategies to control the phase behavior by introducing a cysteine residue to oleosin, a protein that does not contain any cysteines. Adding a cysteine residue to the N-terminal hydrophilic region of oleosin results in phase separation at a lower protein concentration and an increase the phase transition temperature. Furthermore, adding a reducing agent, beta-mercaptoethanol, to phase-separated, cysteine-containing oleosin rapidly dissolves the droplets. We demonstrate two strategies to precisely control the transition temperature of cysteine-containing oleosin. First, the transition temperature is tuned by varying the location of the cysteine in the oleosin backbone. Second, the transition temperature is tuned by blending the parent cysteine-less molecule with the cysteine containing mutant. These methods provide novel ways to control protein droplet formation and dissolution. This work will have applications as a system for the release of molecules with sensitivity to reducing conditions and as an engineered mimic of membrane-less organelles in synthetic protocells.