Date of Award

8-2025

Document Type

Thesis

Degree Name

Master of Science (MS)

College/School

College of Science and Mathematics

Department/Program

Chemistry and Biochemistry

Thesis Sponsor/Dissertation Chair/Project Chair

Eli Il-Hyung Lee

Committee Member

Nina Goodey

Committee Member

Jaclyn Catalano

Abstract

Protein droplets, also referred to as condensates, are membrane-less organelles formed through a biophysical process known as liquid-liquid phase separation (LLPS). These structures play an indispensable role in a wide range of cellular functions, including compartmentalizing biochemical reactions, regulating gene expression, and responding to cellular stress. Unlike traditional membrane-bound organelles, these condensates form dynamically and reversibly within the cytoplasm or nucleus, without the need for lipid membranes. The formation of these protein droplets is affected by several physicochemical factors, including pH, temperature, ionic strength, concentration of biomolecules, and specific molecular interactions such as electrostatics and hydrophobic forces. LLPS results in the demixing (the separation of substances within a mixture) of components into dense droplet phases surrounded by dilute solutions, enabling rapid and localized biochemical activity. These droplets exhibit fluid-like properties and are stabilized through weak, multivalent interactions and surface tension that arises from differential charge distributions within the condensate. Abnormal behavior may result in the transition of fluid droplets into solid-like or gel-like aggregates, contributing to the development of various diseases. These include cancer and neurodegenerative disorders such as Alzheimer’s, Parkinson’s, and Huntington’s diseases, where misregulated protein droplet disrupts cellular homeostasis and protein quality control. Researchers around the world are actively exploring this topic, aiming to uncover the underlying mechanisms behind protein droplet formation through LLPS. They're also working to identify the specific factors that might promote or enhance this process, in hopes of better understanding how these droplets function in both healthy and diseased cells. The use of charged polyelectrolytes, such as poly-L-lysine (PLL) and poly-L-glutamic acid (PLE), which carry positive and negative charges, respectively, provides an effective and controllable way to generate liquid-like condensates through LLPS. The system serves as a powerful experimental model, allowing us to closely observe the behavior of protein droplets under a microscope. By studying these condensates, valuable insights can be gained into how protein droplets form, how they interact with one another, and how they evolve or age over time within different environments. This helps deepen our understanding of the complex dynamics that govern cellular organization and dysfunction. Beyond serving as a model system, the electrostatic interactions between PLL and PLE have demonstrated potential in various biomedical applications. For instance, these interactions can be harnessed to develop advanced drug delivery systems where therapeutic agents are encapsulated within droplets for controlled release. They are also promising in tissue engineering, where phase-separated materials can support cell growth and tissue regeneration. Investigating how different factors, such as concentration, charge ratio, aging time, and external stimuli, affect the stability and functionality of protein droplets is crucial for developing therapeutic strategies. In particular, understanding the role of charged polymers in modulating phase behavior may help in designing interventions to prevent or reverse disease-associated aggregation. Continued research into LLPS and synthetic droplet models not only enriches our understanding of fundamental cell biology but also holds promise for innovative applications in biotechnology and medicine. These insights may pave the way for novel approaches to treat diseases rooted in phase separation abnormalities while opening exciting new frontiers in synthetic biology and materials science.

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