Abstract
Recent discoveries in liquid-liquid phase separation (LLPS) reveal that disordered proteins’ phase separation is promoted by crowding agents, specifically polymers like PEG, dextran and Ficoll. Despite being traditionally considered biologically inert, these polymers can reach high concentrations in aqueous solutions, making them valuable mimics of cellular crowding. However, it’s essential to note that PEG and dextran can also undergo phase separation via segregation, suggestion potential interactions with disordered proteins. In this thesis, the impact of crowders on LLPS will be explored in the aim to create a more physiologically relevant environment for studying model condensates. A nucleolus model was created by mixing the protein nucleophosmin-1 with ribosomal RNA, and a stress granule model was achieved by using the protein G3BP1. By comparing various synthetic crowders such as PEG and dextran, we quantify crowder partitioning and condensate viscoelasticity. Our results underscore that crowding agents are not inert; they play pivotal roles through association and segregation in driving LLPS.
