Defense Date
2026
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Chemistry
First Advisor
Brian Fuglestad
Abstract
Peripheral membrane proteins (PMPs) are a unique class of aqueous proteins which bind to the membrane reversibly for functionality. These proteins play a vital role in many biological processes and mechanisms, making them of high interest. Current technologies and methodologies limit understanding of the membrane-bound structure and function of these proteins in high-resolution. NMR is the most optimized method for observing these small, flexible PMPs, further technology development is needed for more advanced exploration in protein-membrane and protein-lipid interactions.
Reverse micelles (RMs) have the ability to encapsulate a PMP in its membrane-bound state. The protein is housed within a nanoscale water core surrounded by lipid surfactants with headgroups facing inwards towards the aqueous phase and the lipid tails facing outwards towards the alkane solvent. While previously developed RMs were shown to capture the membrane adhered states of PMPs, their formulations do not represent the lipid diversity or the compositions native membranes. This presents the need for new formulations of RMs to fully explore PMP-membrane interactions. Formulating native RMs (nRMs) from lipid extracts, including soybean lecithin, porcine brain, and bovine heart, expand the ability to study membrane-associated proteins in a diverse lipid context. Our newly developed formulations have consistent characteristics expected of RMs, confirmed by DLS and cryo-EM. These formulations successfully encapsulated three PMPs in their membrane-bound state. RMs have been further developed to mimic the complex phospholipid headgroup composition of three cytosolic facing membrane leaflets. Biologically-inspired RMs include cytosolic-facing plasma, mitochondrial, and rough endoplasmic reticulum membrane mimics. Using the plasma membrane RM, sterol carrier protein 2 (SCP2) is captured, for the first time, in its membrane-bound state within its native environment.
Our PMP of interest, glutathione peroxidase 4 (GPx4), is the primary enzyme responsible for reducing lipid hydroperoxides through a glutathione coupled reaction within the cellular membrane, preventing ferroptosis. GPx4 is essential for cell homeostasis and is currently a high-profile cancer target for inhibition. Unfortunately, most of the current research occurs in the soluble state, not in its functional, membrane-bound state. Understanding the functional relationship between lipids and GPx4 will elucidate its full mechanism. Through lipid overlay assays, phosphorylated phosphatidylinositol (PIP) lipids showed an increased affinity for GPx4, comparable to or exceeding known anionic lipid binders. Further NMR experiments, both aqueous and with a membrane model confirmed higher affinity binders of PIP lipids and PIP lipid headgroups, increasing with the number of phosphorylations. The chemical shift perturbations (CSPs) calculated from the NMR data begins to map where PIP lipid headgroups bind to GPx4. The binding site is localized proximal to the cationic membrane interacting site which helps position lipid tails for GPx4 to reduce hydroperoxides. Crystallography further confirms the headgroup binding site while elucidating a possible structural mechanism of GPx4 function. Furthermore, work has been completed to understand the electrostatic interactions of a single point mutation of GPx4 (R152H); it significantly weakens the electrostatic interactions that GPx4 natively has with the membrane, reducing the functionality of GPx4. In order to fully understand the catalytic regeneration of GPx4 with a glutathione coupled reaction, glutathione was bound to GPx4, and preliminary data suggests that GPx4 is not likely to be membrane bound when glutathione is attached. Overall, this work represents significant strides in understanding membrane-bound proteins, both functionally and conformationally. The work completed with GPx4 provides an advanced foundational understanding for lipid binding and affinity towards GPx4, allowing further elucidation of membrane-bound GPx4 and inhibition in future works.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
4-28-2026