ORCID
http://orcid.org/0000-0001-9082-1272
Date of Award
Summer 8-15-2020
Degree Name
Doctor of Philosophy (PhD)
Degree Type
Dissertation
Abstract
Proteins, one of the most fundamental biomolecules, adopt unique higher order structures (HOS) to enable diverse biological functions. Deciphering protein HOS is crucial to gain deeper insights of their working mechanisms and to develop biotherapeutics. Mass spectrometry (MS)-based approaches evolved rapidly in the past 30 years and are now playing critical roles in protein HOS characterization. One of those approaches is MS-based footprinting whose principle is to map the solvent accessible surface area (SASA) to deliver structural information. Protein footprinting can be achieved by reversible labeling, e.g., hydrogen-deuterium exchange (HDX), and by irreversible labeling using radical-based reagents or other targeted labeling reagents. Irreversible labeling such as fast photochemical oxidation of protein (FPOP) and cross-linking (XL) delivers the information of the reactive amino acid side chains, whereas HDX allows the analysis of the backbone amides. Information from the two aspects are different yet complementing to each other. In Chapter 1, the two MS-based footprinting methodologies are reviewed and discussed in detail including the fundamental, history, and recent applications.
In the second section, development of the irreversible radical footprinting is the primary focus, specifically the elaboration of FPOP platform. Chapter 2 describes the generation and evaluation of a new radical reagent, the carbonate radical anion, on the FPOP platform; a radical that can selectively label methionine and aromatic residues, complementing others radical reagents in the footprinter “toolbox”. Chapter 3 demonstrates a novel way of elaborating FPOP platform that is to follow protein unfolding by coupling two lasers together: one to induce protein conformational changes by pH jump and the other to label protein with radical reagents. A time delay between the two lasers enables the characterization of the dominant protein conformations at different stages of unfolding.
The third section emphasizes the integration of several footprinting approaches as well as computational methods for comprehensive analysis of protein HOS. In Chapter 4 and 5, HDX, XL-MS and molecular docking are combined to determine protein-protein binding interfaces and to map epitope/paratope of an antigen-antibody complex, respectively. In particular, Chapter 4 discusses the potential of using HDX to adjudicate candidate docking models for quaternary structure elucidation. Besides molecular docking, homology modeling in combination with XL-MS is also a successful marriage to decipher protein structures, an example is demonstrated in Chapter 6. Restraints derived from cross-links help modify and validate a predicted structure of phycolisome, contributing to the first proposed architecture of the protein complex in cyanobacteria.
The six chapters combine to demonstrate the development and application of MS-based footprinting in protein HOS characterization. Given the effectiveness and powerfulness of these methods, significant contributions by MS-related approaches are well to be expected in the field of structural proteomics
Language
English (en)
Chair and Committee
Timothy Wencewicz Michael Gross
Committee Members
Meredith Jackrel, Joseph A. Fournier, Gaya K. Amarasinghe,
Recommended Citation
Zhang, Mengru, "Development and Application of Mass Spectrometry-Based Approaches for Protein Higher Order Structure Analysis and Protein-Protein Interaction Characterization" (2020). Arts & Sciences Electronic Theses and Dissertations. 2357.
https://openscholarship.wustl.edu/art_sci_etds/2357