Date of Award
Ph.D. in Pharmaceutical Sciences
University of Mississippi
Structural biology plays an important role in modern molecular biosciences. High-resolution hydroxyl radical protein footprinting (HR-HRPF) is a mass spectrometry (MS) based method for analyzing protein topography at a spatial resolution from peptide up to the individual amino acids. Fast photochemical oxidation of protein (FPOP) is an HRPF technique that generates hydroxyl radicals by UV laser-induced photolysis of hydrogen peroxide. FPOP coupled with MS technology has successfully elucidated protein structure information, protein-protein interaction, and protein allosteric or conformational change. Even though FPOP coupled MS has quickly had a large impact on the field of biopharmaceutical analysis, serious limitations still remain. Membrane protein structural analysis by FPOP is challenging due to reports of low oxidative coverage on membrane proteins. Based on the current understanding of hydroxyl radical reactivity of lipids and other amphiphiles, it was believed that the membrane environment acted as a hydroxyl radical scavenger which decreased the effectiveness of hydroxyl radical doses and resulted in low protein oxidation. Chapter Ⅱ has comprehensively studied the mixed cellular membrane with water soluble proteins myoglobin and observed no significant difference with or without the cellular membrane, while the extracellular loop of an integral membrane protein showed greatly reduced oxidation as compared to a soluble version of the same peptide. To investigate the causes of reported poor membrane protein oxidation, we synthesized a radical dosimeter tethered to a Triton X-series amphiphile and used it to quantitatively measure oxidation when fully solvent exposed near the membrane versus in bulk solution. The result showed clearly increased radical scavenging near the micelle surface, probably due to the high local concentration of the organized amphiphile. The study from Chapter Ⅱ contributes to the understanding of the mechanism of HRPF of membrane proteins and provides insight for membrane protein study in a different direction.
Protein-protein and protein-carbohydrate interactions are also extensively studied in pharmacological analysis to understand the protein binding site and protein conformational or allosteric changes upon interaction. Chapter Ⅲ shows using HRPF to understand the interaction interface between cation-independent mannose 6-phosphate receptor (CI-MPR) with a glycoprotein plasmin precursor plasminogen. We used FPOP coupled with MS to determine the changes in solvent accessibility for free CI-MPR versus plasminogen bound CI-MPR. The result from this study identified both protection and exposure on the protein surface area due to the binding of specific domains of the CI-MPR. This information helps us understand the binding-induced change on other domains of CI-MPR and further investigate the downstream signaling formation.
Another topic of urgent need in structural pharmacology is to understand protein conformers in a dynamic and conformationally heterogeneous system. This has long been a challenge for dynamic systems such as protein aggregation and dynamic post-translational modification. In such systems, FPOP results only can reflect the average conformation of all the protein conformers. To overcome this obstacle, in Chapter Ⅳ we describe a novel method we developed by coupling ion exchange HPLC with a flash oxidation system (IEX LC-FOX) to successfully structurally characterize three different phosphoproteoforms of the ovalbumin (OVA) in a dynamic system during a reaction with alkaline phosphatase (AP). From this study, we were able to identify specific structural changes that occur upon dephosphorylation of individual phosphoSer residues, and validate our findings against both molecular dynamics simulations and previous biophysical studies of OVA phosphorylation.
In summary, this dissertation expands the use and understanding of FPOP’s ability to elucidate the HOS of proteins and proteoforms. These studies will help researchers overcome challenges in biopharmaceutical developing to make drugs faster and safer.
Cheng, Zhi, "Development of Hydroxyl Radical Protein Footprinting for Challenging Systems" (2022). Electronic Theses and Dissertations. 2425.
Available for download on Thursday, February 08, 2024