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Vennela Mullangi - PhD Dissertation.pdf (2.24 MB)

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Development and Application of Histidine Hydrogen Deuterium Exchange Mass Spectrometry.

Mullangi, Vennela, Dr
http://rave.ohiolink.edu/etdc/view?acc_num=csu1388959354

Abstract Details

2013, Doctor of Philosophy in Clinical-Bioanalytical Chemistry, Cleveland State University, College of Sciences and Health Professions.
We recently developed a structural mass spectrometry method termed “histidine hydrogen-deuterium exchange mass spectrometry (His-HX-MS)”. The method determines the hydrogen exchange (HX) rates at imidazole Ce1-hydrogen of histidine residues in proteins using mass spectrometry. The HX rate of the imidazole Ce1-hydrogen varies depending on the degree of exposure to bulk solvent, thus offering a sensitive measure of the solvent accessibility. This method can also determine the pKa values of histidine residues by carrying out an ordinary acid-base titration experiment (pH versus HX rate). Since the acid dissociation constant (pKa) of a histidine imidazole group changes in response to the adjacent ionizable group(s), the pKa value is an useful indicator of the electrostatic environment of the histidine residue. Thus the two parameters (HX rates and pKa values) are useful for understanding the structure-function relationship and dynamic nature of the protein. We have demonstrated the usefulness of the method to study structural changes of proteins induced by binding to ligands and other proteins. In the first study, we investigated the missing knowledge that is required for the quantitative description of solvent accessibility. The measure of solvent accessibility expressed as protection factor value is an independent value and can be compared with the values of other imidazole groups in the same protein or in other proteins. The second study established an analytical methodology using His-HX-MS to study protein stability using RNase A as a model. The method was also applied to study the stability of a protective antigen of anthrax toxin and protective antigen bound to cellular receptor. Concluded from the results of this study, two significant advantages were proposed of this method opposed to the conventional methods such as fluorescence spectroscopy and circular dichroism (CD) spectroscopy; 1) ability to monitor the stabilities of multiple sites within a protein simultaneously, and 2) the method does not require pure proteins because mass spectrometry is used as a mean to monitor histidine containing peptides. The two advantages will allow us to study, for example, the stability of individual domains in multi-domain proteins in complex protein mixtures like yeast cell lysate.
David Anderson, Dr. (Committee Chair)
Masaru Miyagi, Dr. (Committee Co-Chair)
Xiang Zhou, Dr. (Committee Member)
Mekki Bayachou, Dr. (Committee Member)
David Ball, Dr. (Committee Member)
135 p.
Analytical Chemistry

Recommended Citations

Citations

  • Mullangi, V. (2013). Development and Application of Histidine Hydrogen Deuterium Exchange Mass Spectrometry. [Doctoral dissertation, Cleveland State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=csu1388959354

    APA Style (7th edition)

  • Mullangi, Vennela. Development and Application of Histidine Hydrogen Deuterium Exchange Mass Spectrometry. 2013. Cleveland State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=csu1388959354.

    MLA Style (8th edition)

  • Mullangi, Vennela. "Development and Application of Histidine Hydrogen Deuterium Exchange Mass Spectrometry." Doctoral dissertation, Cleveland State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=csu1388959354

    Chicago Manual of Style (17th edition)

csu1388959354
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© 2013, all rights reserved.
This open access ETD is published by Cleveland State University and OhioLINK.