Mass Spectrometry for Determination of Conformation and Dynamics of Proteins and Structure and Biosynthesis of Bacterial Peptidoglycan

Mass spectrometry: MS) has emerged as an important tool for analyzing and characterizing large biomolecules. In this dissertation, two aspects of the development and application of MS-based approaches are presented; they include: 1) protein conformation and folding dynamics: in Chapters 2 to 5) and bacterial peptidoglycan: PG) structure and biosynthesis: Chapters 6 to 8). Chapter 1 serves as the introduction for both aspects. Part I of the dissertation focuses on the development of analytical methods combining fast photochemical oxidation of proteins: FPOP) and mass spectrometry analysis. In chapter 2 to 4, we discuss protein folding with sub-millisecond time resolution by a new pump/probe procedure. Perturbations in protein structure are by temperature jump of the protein solution, followed by fast photochemical oxidation of proteins: FPOP) as the probe. The hydroxyl radical lifetime was predicted by a dosimeter experiment: Chapter 2). The T jump-induced folding constant was measured at the global protein level: Chapter 3), and the residue level detail was revealed by proteolysis and liquid chromatography-mass spectrometry: LC-MS): Chapter 4). Chapter 5 discusses the development of a new FPOP reagent, iodobenzoic acid, and its application on studying conformational differences between apo- and holo- proteins. Part II of the thesis discusses the development and application of MS-based methods to investigate bacterial peptidoglycan. Chapter 6 focuses on the methodology of the bottom-up MS to characterize the fine structure of <italic>enterococcus faecium</italic>: <italic>E. faecium</italic>) peptidoglycan. Furthermore, we developed a time-dependent isotopic labeling strategy and applied it to <italic>E. faecium</italic> during the cell wall growth to determine quantitatively the percentage of heavy isotope incorporation into different muropeptides through peptidoglycan growth cycles, discussed in chapter 7. The results are important for understanding tertiary structure and designing novel drugs for antibiotic-resistant pathogens. In chapter 8, we applied the above approaches to investigate methicillin-resistant staphylococcus aureus: <italic>S. aureus</italic>) and its <italic>fem</italic>-mutants. We emphasize the peptidoglycan composition, fine structures, and biosynthesis.

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Chemistry

Year dc:date.available

2011

Author dc:creator

• Chen, Jiawei

Contributors dc:contributor

• Michael Gross

• Biophysics
• Analytical chemistry
• Biochemistry
• LC/MS
• Mass spectrometry
• Peptidoglycan
• Protein

Language dc:language

English (en)

DOI

https://doi.org/10.7936/K7W093ZH

OAI identifier oai:identifier

oai:openscholarship.wustl.edu:etd-1561