Engineering Stimuli-Responsive Protein Phase Separations for Protein Stabilization, Modulation of Protein Activity, and Intracellular Protein Delivery

Proteins and peptides represent a unique class of biological materials, capable of self-assembling into higher-order structures and participating in multiple types of phase separation. Recruitment of proteins into separate phases or small-scale self-assemblies has been leveraged to produce unique behaviors in peptides, with applications in intracellular protein delivery, increasing enzyme catalytic activity, and protein separation and purification. Proteins are chemically diverse and highly modifiable, both through post-translational modification of amino acid residues and through modification of the primary peptide sequence. Here, we use the unique chemical and genetic modifiability of proteins to control protein self-assembly, phase separation, and tertiary structure in several model systems. In the first of these systems the gene editing enzyme, cas9 is modified to facilitate encapsulation in protein-synthetic polymer polyelectrolyte complex (PEC) micelles. These micelles were designed for endosomal uptake and then subsequent disassembly at mildly acidic pH and in the presence of endosomal proteases to facilitate endosomal escape of the cas9 protein cargo. In the second system, we designed an intrinsically disordered peptide to form a stimuli-responsive, entirely peptide PEC micelle system, capable of encapsulating a supercationic protein. The phase behavior of the peptide PEC micelles was characterized with respect to salt concentration, positive charge fraction, and temperature. Finally, an enzymatic protein was conjugated to a temperature- and light-responsive polymer. The resulting bioconjugate was purified and shown to undergo light- and temperature-induced changes in the phase behavior