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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Investigation of Metalloproteins Utilizing High Resolution Mass Spectrometry

Wu, Zhaoxiang 2010 May 1900 (has links)
Copper ions (Cu⁺, Cu²⁺) play important roles in many biological processes (i.e., oxidation, dioxygen transport, and electron transfer); many of the functions in these processes result from copper ions interacting with proteins and peptides. Previous studies using matrix assisted laser desorption/ionization (MALDI) mass spectrometry (MS) have shown that Cu⁺ ions preferentially bind to electron rich groups in gas phase (i.e., N-terminal amino group, the side-chains of lysine, histidine and arginine). For peptides with more than one Cu⁺ ligand, the interaction between Cu⁺ ions and ligands is described in terms of competitive binding; however, Cu⁺ coordination chemistry for multiple Cu⁺-containing proteins and peptides in gas phase is still not fully understood. In addition, no studies on the fragmentation chemistry for multiple Cu⁺-binding peptides, such as [M + 2Cu - H]⁺ ions, have been reported. The synthesized dinuclear copper complex (alpha-cyano-4-hydroxycinnamic acid (CHCA) copper salt (CHCA)₄Cu₂) enhances the ion abundances for [M + xCu - (x-1)H]⁺ (x = 1-6) ions in gas-phase when used as a MALDI matrix. Using this matrix we have investigated site-specific copper binding of several peptides using fragmentation chemistry of [M + Cu]⁺ and [M + 2Cu - H]⁺ ions. The fragmentation studies reveal that the binding of a single Cu⁺ ion and two Cu⁺ ions are different, and these differences are explained in terms of intramolecular interactions of the peptide-Cu ionic complex. The competitive Cu⁺ binding to C-terminus (i.e., amide, carboxyl, methyl ester) versus lysine, as well as cysteine (SH/SO₃H) versus arginine (guanidino), was also examined by MALDI MS and theoretical calculations (Density Functional Theory (DFT)). For example, results from theoretical and experimental (fragmentation reactions) studies on [M + Cu]⁺ and [M + 2Cu - H]⁺ ions suggest that cysteine side chains (SH/SO₃H) are important Cu⁺ ligands. Note that, the proton of the SH/SO₃H group is mobile and can be transferred to the arginine guanidino group. For [M + 2Cu - H]⁺ ions, deprotonation of the -SH/SO₃H group is energetically more favorable than that of the carboxyl group, and the resulting thiolate/sulfonate group plays an important role in the coordination structure of [M + 2Cu - H]⁺ ions.
2

PROBING GAS-PHASE PEPTIDE STRUCTURE AND PROTEIN-PROTEIN INTERACTIONS USING MASS SPECTROMETRIC TECHNIQUES

Perkins, Brittany January 2009 (has links)
Presented in this dissertation are studies on the gas-phase structural features of peptides and peptide fragment ions using mass spectrometry (MS), hydrogen/deuterium (H/D) exchange, infrared multiphoton dissociation (IRMPD) spectroscopy, and computational modeling. Additional studies are presented on the mechanism of hydrogen/deuterium exchange using a model amino acid system. The application of chemical cross-linking to investigate the interaction between two proteins, LexA and RecA, is also presented. Gas-phase structural features can be probed using a number of techniques, and several of the studies presented in this dissertation involve the use of gas-phase H/D exchange. Although the basic mechanism for exchange has been determined, the factors that affect the rate and extent of exchange are not well understood. A computational modeling study of the exchange behavior of asparagine and its methyl ester demonstrated that exchange will occur preferentially at sites of more similar basicity. The distinctive exchange behavior of a model histidine-containing pentapeptide, HAAAA, prompted further studies into the structural features that result in five fast exchanging hydrogens and one slower exchange. Peptide analogues were used to identify the sites of exchange, and IRMPD spectroscopy combined with computational modeling indicated that exchange may occur because interaction with water at those sites results in lower energy structures compared to the other sites. Structural studies were also performed to determine whether the b₂⁺ ion from HAAAA is an oxazolone or diketopiperazine. Although the IRMPD spectrum matched that of a diketopiperazine, H/D exchange and fragmentation studies revealed the presence of both a diketopiperazine and oxazolone structure. Protein-protein interactions perform a vital role in regulating cellular processes. Despite extensive mutational analysis, the binding interaction between LexA and RecA, two proteins involved in the SOS response, is unclear. Chemical cross-linking experiments were undertaken to help target future mutational studies, and these studies identified two possible interactions. The first potential binding interaction is located in the cleft of RecA, and the second interaction may be caused by a LexA dimer binding across the RecA helical groove. The presence of two different binding interactions suggests that LexA may have redundant binding modes for RecA interaction.
3

The Study of Inter and Intramolecular Interactions in Gas Phase Protein Ions by Electron Transfer Dissociation

Browne, Shaynah J 01 January 2012 (has links) (PDF)
Mass spectrometry (MS) is emerging as an important tool for studying protein and protein complexes. When applying this tool, it is important to understand and investigate whether some of the intramolecular and intermolecular interactions of proteins in solution and are maintained in the gas phase. To investigate if some of these interactions are maintained in the gas phase, we develop and use a method in which the electron-transfer dissociation (ETD) spectra of native proteins are compared with spectra from ETD followed by low amplitude collisional induced dissociation (CID). From these experiments, we find that some intramolecular interactions from solution are maintained in the gas phase for ubiquitin and beta-2-microglobulin (β2m). However, using these approaches, cytochrome c’s structure in the gas phase appears to be quite different than its structure in solution. We also investigated if ETD spectra of intact protein complexes reflect contact site information in these complexes

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