Defense Date
2024
Document Type
Thesis
Degree Name
Doctor of Philosophy
Department
Chemistry
First Advisor
Brian Fuglestad
Abstract
Fatty acid binding proteins (FABPs) are pivotal in orchestrating lipid trafficking within cells. This dissertation aims to reveal the mechanism of action of adipocyte FABP (FABP4) by meticulously detailing steps for producing uniformly isotopically labeled protein, understanding dynamics and structural changes in aqueous conditions, analyzing protein-membrane interaction with membrane mimicking models, and revealing its membrane-bound state through novel NMR structural elucidation. Chapter 1 provides an overview of FABP4's indispensable role in lipid trafficking, emphasizing its significance in maintaining cellular homeostasis and metabolic regulation. Chapter 2 delves into the optimization of FABP4 protein expression and proposes a high yield recovery and reproducible delipidation protocol, ensuring the quality of protein samples for subsequent studies throughout the dissertation. Chapter 3 explores aqueous NMR assignments and studies of FABP4, elucidating possible structural changes between the Apo and Holo states in aqueous conditions, despite the indistinguishable backbone structure. Chapter 4 investigates the elusive interactions of FABP4 with membranes, utilizing a diverse portfolio of membrane mimicking models to simulate conditions where lipids are delivered into the protein cavity from target membranes. Finally, Chapter 5 unveils the groundbreaking revelation of FABP4's membrane-bound state using membrane-mimicking reverse micelles (mmRMs), providing, for the first time, an NMR structure that not only elucidates a never-before-seen membrane-bound state of FABP4 but also underscores its crucial role in modulating lipid trafficking processes within cells. These findings offer crucial insights into the mechanism of misregulated lipid metabolism-related diseases, ultimately suggesting alternate therapeutic designs for FABPs in treating metabolic disorders, obesity-related diseases, and cardiovascular diseases.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
5-10-2024