(741a) Controlled Liquid-Liquid Phase Separation of Recombinant Oleosin

Protein engineering enables the creation of materials with designer functionality and responsiveness. In this work, we designed a protein that phase separates into liquid droplets upon oxidation of a cysteine residue. The phase transition temperature can be tuned via the location of the cysteine residue in the protein backbone. Our work is based on the naturally occurring plant protein, oleosin. The protein is amphiphilic, with distinct hydrophilic and hydrophobic domains: an N-terminal hydrophilic segment, a highly hydrophobic central core, and a C-terminal hydrophilic segment. From wild type oleosin, a truncated version was engineered that reduced the hydrophobic core from 87 amino acids to 30 and added 5 glycines. Circular dichroism indicates that this mutant has less secondary structure than the wild type and approximately 65% of the protein is disordered. A single amino acid mutation was made to introduce a cysteine residue in the hydrophilic arm. Oxidation of this residue results in a dimer. Placing the cysteine closer to the N-terminus raises the transition temperature. The phase behavior of the protein can be eliminated by reducing the protein. Because the transition temperature varies with the location of the added cysteine, this protein provides a novel way to control protein liquid droplet formation.