PROJECT SUMMARY Current research proposes protein phase separation as a key mechanism underlying the formation of liquid-like biomolecular condensates, which play an integral role in numerous cellular processes and age-related disorders. The interior of a living cell is a highly crowded environment, therefore, to fully comprehend the phase behaviors and biological functions of proteins, it is critical to consider the densely populated environment of cells, where proteins coexist with a wide variety of molecules possessing diverse physical and chemical properties. To this end, researchers have utilized various molecules as crowding agents to study protein phase behavior in this context. While the effects of crowding on proteins have been studied to some extent, many open questions remain to be addressed. The long-term goal of the proposed research is to unravel the mechanism underlying the phase separation of proteins in living cells. Our previous research explored the impact of two types of crowding agents on a model protein/polymer system, revealing that they lead to distinct phase-separated complexes due to their different effects on individual protein and polymer molecules. Therefore, based on the previous results, our central hypothesis is that the observed macroscopic crowding phenomena are directly linked to how the crowding agents affect the conformation and behavior of individual protein and polymer molecules at the molecular level. The overall objectives of this study are to enhance our understanding of the changes in conformation and intermolecular interactions that regulate protein phase behavior in crowded environments, and how these changes impact the properties of the phase separated proteins. The central hypothesis and objectives of this application will be tested and attained by pursuing four specific aims: 1) investigate the effects of crowing on the phase transition of a model protein/polymer system; 2) characterize the physical properties of the phase separated protein/polymer complexes; 3) measure the conformation of individual protein and polymer molecules in different crowding environments; 4) probe the effects of crowding on the protein- protein and protein-polymer interactions. The proposed research is anticipated to have a significant positive impact, as a detailed understanding on the protein phase behavior in crowded environments will shed light on the formation, function, and fate of biomolecular condensates. The contribution is significant because it marks the first step in a series of investigations expected to lead to a comprehensive understanding of protein phase behavior in crowded environments at the molecular level.