Effects of molecular and macromolecular crowding agents on protein/polymer complex coacervation

Complex coacervation refers to the liquid-liquid phase separation (LLPS) due to the strong associative interactions between charged macromolecules. In recent years, the study of complex coacervation has been used to explain the formation of membraneless organelles (MLOs) in living cells. MLOs are believed to be formed by the LLPS of protein and nucleic acids within the cellular environment. However, most of the studies conducted to understand the formation of MLOs lack the compositional richness to properly mimic the crowded cellular environment where the MLOs are believed to form. In this context, we aim to investigate the effects of crowding on a model protein/polymer complex coacervate system. To introduce such crowdedness into our model system, sucrose, and polyethylene glycol (PEG) have been used as molecular and macromolecular crowder, respectively. Our findings demonstrated that sucrose, as a molecular crowder, had minimal effects on the physical properties of the complex coacervates, the presence of PEG resulted in coacervates with increased density, droplet size, and protein/polymer content. Furthermore, a compact internal structure with a closely packed arrangement of proteins was observed within the coacervate prepared in the presence of PEG. These variations in coacervate properties can be attributed to the impacts of crowders on individual macromolecules, including the conformation of model polymer, and non-specific interactions among model protein molecules. Furthermore, our findings show that sucrose gets distributed in both coacervate and dilute phases, whereas PEG was found only in the dilute phase. Collectively our results shed light on understanding the effects of crowding on complex coacervation, while highlighting the formation and properties of coacervates in the context of MLOs.