Date of Award
Winter 12-15-2018
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
Membrane proteins are essential in many cellular processes and represent ~60% of all current drug targets. Due to technical limits, membrane proteins of various types were not studied extensively in the past and the biochemistry and functionality of them remain unclear. The structural biology methodologies require pure isolated protein samples for us to resolve their structure and study their biochemical functions. For such in vitro studies, however, membrane proteins often become unstable when isolated from their native lipid bilayer environment. To overcome the challenge, I employed a novel methodology of solubilizing membrane proteins in solution without detergent. I reconstituted ferroportin, an iron exporter, into saposin A lipoprotein discs, which provide a phospholipid bilayer environment for stabilizing the ferroportin. Those ferroportin picodiscs samples were further used for mass spectrometry footprinting experiments. Based on the analysis, different footprinting methods are an efficient and extensive probe of specific residues over the entire sequence of ferroportin. Footprinting results by fast photochemical oxidation of proteins (FPOP) show that the extramembrane regions of ferroportin in picodiscs are extensively oxidized by free hydroxyl radicals, whereas transmembrane regions are more protected, suggesting the native structure of ferroportin is retained throughout the labeling process. In contrast, footprinting by NEM, a membrane-permeable reagent, showed extensive labeling of cysteines in both the transmembrane (hydrophobic) regions and extramembrane (hydrophilic) regions. HDX digestion of ferroportin was done in ferroportin picodiscs and a ~92% sequence coverage was achieved for footprinting. Those results demonstrate saposin picodiscs to be a feasible tool for membrane protein studies with footprinting mass spectrometry methods.
Because membrane proteins remain a challenge for experimental studies, I performed molecular dynamics (MD) simulation for studying membrane proteins and started a platform specifically for simulating membrane protein systems. I studied a variety of membrane protein systems including VKOR-warfarin binding, and HMGCR-UBIAD1 complex. Such work has provided mechanistic models to illustrate the binding protein-protein interactions and protein-ligand binding mechanisms in a phospholipid bilayer membrane.
Those work are described in detail across all chapters. Chapter 1 serves as in introduction and overview of the MS and MD for membrane protein studies. Chapter 2 focuses on combining MS and MD to study human VKOR, and its binding mechanisms of VKOR-warfarin. Chapter 3 focuses on the preparation of the materials required for building saposin A picodiscs, and reconstituting membrane proteins into picodiscs. Chapter 4 and 5 present mass spectrometry studies and approaches of studying ferroportin picodiscs and its interaction with hepcidin, an inhibitive ligand of ferroportin. The manuscript of the work presented in those chapters is under preparation for publication. Chapter 6 describes a pure MD simulation study on I performed for building a UBIAD1-HMGCR binding model. Both UBIAD1 and HMGCR were generated with atomic models at best authority, with UBIAD1 generated as a homology model and HMGCR as a hybrid homology model. The manuscript of the work presented in Chapter 6 is finished and under preparation for publication.
Language
English (en)
Chair and Committee
Weikai Jianmin . Li Cui
Committee Members
Eric Galburt, Michael Gross, Peng Yuan,
Recommended Citation
Zhou, Fengbo, "Combining Footprinting Mass Spectrometry and Molecular Dynamics Simulation for Structural Studies in Membrane Proteins" (2018). Arts & Sciences Electronic Theses and Dissertations. 1686.
https://openscholarship.wustl.edu/art_sci_etds/1686
Comments
Permanent URL: https://doi.org/10.7936/52k5-gf85