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dc.contributor.advisorBrodbelt, Jennifer S.en
dc.creatorCammarata, Michael B.en
dc.date.accessioned2016-07-19T21:30:46Z
dc.date.available2016-07-19T21:30:46Z
dc.date.issued2014-12en
dc.date.submittedDecember 2014
dc.identifierdoi:10.15781/T26688J7Wen
dc.identifier.urihttp://hdl.handle.net/2152/39140en
dc.description.abstractUltraviolet photodissocation (UVPD) mass spectrometry was used for high mass accuracy top down characterization of two proteins labeled by the chemical probe, S-ethylacetimidate (SETA), in order to evaluate conformational changes as a function of denaturation. The SETA labeling/UVPD-MS methodology was used to monitor the mild denaturation of horse heart myoglobin by acetonitrile, and the results showed good agreement with known acetonitrile and acid unfolding pathways of myoglobin. UVPD outperformed another ion activation method, electron transfer dissociation (ETD), in terms of sequence coverage, allowing the SETA reactivity of greater number of lysine amines to be monitored and thus providing a more detailed map of myoglobin. This strategy was applied to the third zinc-finger binding domain, domain C, of PARP-1 (PARP-C), to evaluate the discrepancies between the NMR and crystal structures which reported monomer and dimer forms of the protein, respectively. The trends reflected from the reactivity of each lysine as a function of acetonitrile denaturation supported that PARP-C exists as a monomer in solution with a close-packed C-terminal alpha helix. Additionally, those lysines for which the SETA reactivity increased under denaturing conditions were found to engage in tertiary polar contacts such as salt bridging and hydrogen bonding, providing evidence that the SETA/UVPD-MS approach offers a versatile means to probe the interactions responsible for conformational changes in proteins. UVPD mass spectrometry was also employed to investigate the structure of holo-myoglobin as well as its apo form transferred to the gas phase by native electrospray. The fragmentation yields from UVPD showed the greatest overall correlation with B-factors generated from the crystal structure of apo-myoglobin, particularly for the more disordered loop regions. Comparison of UVPD of holo- and apo- myoglobin revealed similarities in fragmentation yields, particularly for the lower charge states (8 and 9+), but those regions involved in harboring the heme group (for the holo form) exhibited significantly lower fragmentation than the apo-myoglobin state. Both holo- and apo-myoglobin exhibited low fragmentation yields for the AGH helical core (reflecting its highest stability).en
dc.format.mimetypeapplication/pdfen
dc.language.isoenen
dc.subjectMass spectrometry
dc.subjectUltra-violet photodissociation
dc.subjectStructural biology
dc.subjectMyoglobin
dc.subjectParp-c
dc.titleDevelopment of top-down methods for evaluating protein structure and protein unfolding utilizing 193 nm ultraviolet photodissociation mass spectrometryen
dc.typeThesisen
dc.date.updated2016-07-19T21:30:46Z
dc.contributor.committeeMemberWebb, Lauren J.en
dc.description.departmentChemistryen
thesis.degree.departmentChemistryen
thesis.degree.disciplineChemistryen
thesis.degree.grantorThe University of Texas at Austinen
thesis.degree.levelMastersen
thesis.degree.nameMaster of Artsen
dc.type.materialtexten


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