(636c) A Study of Recombinant Fusion Proteins Self-Assembly in Macromolecularly Crowded Conditions

A bottom-up construction of biomimetic artificial cells has been developed in diverse forms to simplify the complexity of living cells whiles also focusing on the structure and functions of biological components. For instance, biochemical processes in living systems occur in macromolecularly crowded conditions. Macromolecularly crowded conditions influence the properties of biological molecules in the solution due to the excluded volume effect and nonspecific intermolecular interactions that exist. Therefore, it is crucial to investigate the behavior of biomolecules in artificial cell platforms under crowded conditions. Self-assembly of the recombinant fusion protein is an effective bottom-up approach for synthesizing various ordered structures such as coacervate or vesicles. We utilize the self-assembly of recombinant fusion proteins, including globular protein and elastin-like polypeptide (ELP), to make artificial cell platforms. Herein, we focus on the effect of polyethylene glycol (PEG), which is a widely used crowding agent in biomimetic systems, on the alteration of protein self-assembled structure. We hypothesized the protein self-assembled structures could be directly affected by the concentration of PEG crowding agents because crowding agents reduce the volume of solvent available for protein building blocks. In addition, PEG can induce the structural transition of protein self-assembly due to interference in protein-protein and protein-solvent interactions at the protein-rich phase interfaces in the solution. To test this hypothesis, we investigate the detailed phase transition behavior of the protein self-assembled structures under PEG crowded conditions at different concentrations by in situ fluorescent microscopy and dynamic light scattering (DLS) measurements. A study of the self-assembly of recombinant fusion proteins in macromolecularly crowded conditions would offer critical information for biological applications of artificial cells.