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.

Cationic polyelectrolytes prevent the aggregation of l-lactate dehydrogenase under unstable conditions.

Unstructured biological macromolecules have attracted attention as protein aggregation inhibitors in living cells. Some are characterized by their free structural configuration, highly charged, and water-soluble. However, the importance of these properties in inhibiting protein aggregation remains unclear. In this study, we investigated the effect of charged poly (amino acids), which mimic these properties, on aggregation of l-lactate dehydrogenase (LDH) and compared their effects to monomeric amino acids and folded proteins. LDH was stable and active at a neutral pH (~7) but formed inactive aggregates at acidic pH (< 6). Adding cationic polyelectrolytes of poly-l-lysine and poly-l-arginine suppressed the acid-induced aggregation and inactivation of LDH under acidic pH values. Adding monomeric amino acids and cationic folded proteins also prevented LDH aggregation but with lower efficacy than cationic polyelectrolytes. These results indicate that unstructured polyelectrolytes effectively stabilize unstable enzymes because they interact flexibly and multivalently with them. Our findings provide a simple method for stabilizing enzymes under unstable conditions.