Interfacial and intrinsic molecular effects on the phase separation/transition of heteroprotein condensates.

Liquid-liquid phase separation (LLPS) and droplet formation by LLPS are key concepts used to explain compartmentalization in living cells. Protein contact to a membrane surface is considered an important process for protein organization in a liquid phase or during transition to a solid or liquid dispersion state. The direct experimental comprehensive investigation is; however, not performed on the surface-droplet interaction and phase transition. In the present study, we constructed simple and reproducible experiments to analyze the structural transition of aggregates and droplets in an ovalbumin (OVA) and lysozyme (LYZ) complex on glass slides with various coatings. The difference in droplet-surface interaction may only be important in the boundary region between aggregates and droplets of a protein mixture, as shown in the phase diagram. Co-aggregates of OVA-LYZ changed to droplet-like circular forms during incubation. In contrast, free l-lysine resulted in the uniform droplet-to-solid phase separation at lower concentrations and dissolved any structures at higher concentrations. These results represent the first phase-diagram-based analysis of the phase transition of droplets in a protein mixture and a comparison of surface-surface and small molecular-droplet structure interactions.