ORCID

http://orcid.org/0000-0003-3198-1588

Date of Award

Winter 1-15-2021

Author's School

Graduate School of Arts and Sciences

Author's Department

Chemistry

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Protein ligand interaction is a fundamental question in biology and biochemistry, and many approaches including X-ray crystallography, nuclear magnetic resonance, cryogenic electron microscopy, mass spectroscopy (MS), infrared spectroscopy, circular dichroism, fluorescence spectroscopy and many others have been applied to address this question. Among these techniques, mass spectroscopy has the advantage of high throughput, low sample amount requirement, and mid-to-high spatial resolution. One of the MS-based approaches is protein footprinting, which utilizes labeling reagents to map the solvent accessible surface of the protein of interest thus deliver structural information. Irreversible labeling is represented by covalent labeling and radical labeling, in which labeling reagents react with amino acid side chains. Reversible labeling, on the other hand, is represented by hydrogen deuterium exchange (HDX), allowing the analysis of protein backbone. This dissertation describes the development of mass spectrometry-based approaches for protein higher order structure analysis, with an emphasize on the characterization of protein-ligand interaction analysis. The dissertation is divided into seven chapters, five of them describe original research. The first chapter introduces the mass spectrometry-based protein footprinting for protein higher order structure analysis, including historical overview, basic principles, major applications, and recent advancements.Chapter 2 and 3 describes a novel mass spectrometry-based method, LITPOMS, which combines ligand titration, fast photochemical oxidation of proteins (FPOP), and mass spectrometry measurements to assess protein ligand binding stoichiometry, binding sites, binding orders, affinities, and allosteric behaviors. The method was first demonstrated by melittin – holo-calmodulin binding, whose binding stoichiometry is 1:1 (Chapter 2). Chapter 3 describes an application of the LITPOMS in characterizing the calcium-calmodulin binding system. As a result, the calcium binding sites, binding orders, site-specific binding affinities, and most importantly the allosteric behavior of calmodulin upon binding with calcium was revealed via a single experiment. Chapter 4 highlights a mechanistic study of FPOP chemistry, through selective 18O labeling coupled with mass spectrometry analysis, and revealed the amino acid-specific oxygen uptake pathways in the FPOP platform and further highlighted the potential of tailoring the FPOP labeling condition to address different biological questions. Chapter 5 and 6 report application of HDX in addressing novel biological questions. Chapter 5 presents a thorough analysis of tetraspanin CD53 and CD81 with their binding partners and revealed the importance of tetraspanin open conformation in facilitating the interaction network. Chapter 6 describes an HDX study of the interaction between Class II lanthipeptide synthetase HalM2 with its partner HalA2-LP. Through a two-temperature HDX workflow, the confidence of binding site assignment increases significantly, especially for weak binding systems. The last chapter highlights the perspective and future work. These chapters combine to demonstrate the scope of developing and adopting mass spectrometry-based approaches to characterize protein-ligand interactions.

Language

English (en)

Chair and Committee

Michael L. Gross

Committee Members

Timothy Wincewicz, Joseph A. Fournier, Meredith Jackrel, Weikai Li,

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