Hydrogen deuterium exchange mass spectrometry for protein-protein interaction and structural dynamics

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dc.contributor Frantom, Patrick A.
dc.contributor Street, Shane C.
dc.contributor Vincent, John B.
dc.contributor O'Donnell, Janis M.
dc.contributor.advisor Busenlehner, Laura S.
dc.contributor.author Singh, Harsimran
dc.date.accessioned 2017-03-01T16:56:57Z
dc.date.available 2017-03-01T16:56:57Z
dc.date.issued 2013
dc.identifier.other u0015_0000001_0001481
dc.identifier.other Singh_alatus_0004D_11717
dc.identifier.uri https://ir.ua.edu/handle/123456789/1944
dc.description Electronic Thesis or Dissertation
dc.description.abstract Hydrogen deuterium exchange mass spectrometry has emerged as an important technique to probe protein structure and conformational dynamics. The rate of exchange of hydrogen with deuterium by the peptide backbone is dependent on the solvent accessibility, extent of hydrogen bonding in secondary structural elements and protein dynamics. The extent and the rate of deuterium incorporation are affected by changes in protein structure, interaction with ligand, protein-protein interaction and environmental factors such as pH and temperature. These conformational changes can be global and/or local. The increase in the mass is used to localize the deuterium incorporation after pepsin digestion of the protein and analysis by electrospray ionization mass spectrometry. In this dissertation traditional HDX-MS and a new deuterium trapping assay were used to probe the interaction sites between E. coli cysteine desulfurase SufS and acceptor protein SufE. SufS and SufE form an important part of the SUF pathway, essential for the biosynthesis of Fe-S clusters under oxidative stress and iron depletion conditions. In addition, SufE is known to stimulate SufS cysteine desulfurase activity, but the mechanism is unknown. The HDX-MS results show that the regions affected by the SufS-SufE interaction are dependent on the catalytic intermediate states of the two proteins. HDX-MS was also used to probe the conformational changes resulting upon persulfuration of SufS of Cys364 in the active site. The persulfuration of SufS not only affected regions in the active site cavity, but also had other conformational changes in more distal regions. Based on our findings a model for the interaction SufS and SufE was proposed. A mechanism for the enhancement of SufS cysteine desulfurase activity upon interaction with SufE was also postulated. In all this work demonstrates that hydrogen deuterium exchange mass spectrometry and the deuterium trapping methodology optimized for this system can be easily and effectively used to study the protein-protein interactions and the accompanying changes in structural dynamics for other proteins. Deuterium trapping was demonstrated to be fast, sensitive and reliable method to deduce the changes in solvent accessibility between two or more states of a protein. Both techniques can easily be applied to large number of protein complexes to determine the regions of interaction as well as gain mechanistic information not available through traditional methods such as X-ray crystallography and NMR.
dc.format.extent 159 p.
dc.format.medium electronic
dc.format.mimetype application/pdf
dc.language English
dc.language.iso en_US
dc.publisher University of Alabama Libraries
dc.relation.ispartof The University of Alabama Electronic Theses and Dissertations
dc.relation.ispartof The University of Alabama Libraries Digital Collections
dc.relation.hasversion born digital
dc.rights All rights reserved by the author unless otherwise indicated.
dc.subject.other Biochemistry
dc.title Hydrogen deuterium exchange mass spectrometry for protein-protein interaction and structural dynamics
dc.type thesis
dc.type text
etdms.degree.department University of Alabama. Dept. of Chemistry
etdms.degree.discipline Chemistry
etdms.degree.grantor The University of Alabama
etdms.degree.level doctoral
etdms.degree.name Ph.D.

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