Unraveling Macro-Molecular Machinery by Mass Spectrometry: from Single Proteins to Non-Covalent Protein Complexes

Persistent Link:
http://hdl.handle.net/10150/195466
Title:
Unraveling Macro-Molecular Machinery by Mass Spectrometry: from Single Proteins to Non-Covalent Protein Complexes
Author:
Cheng, Guilong
Issue Date:
2007
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Presented in this dissertation are studies of protein dynamics and protein/protein interactions using solution phase hydrogen/deuterium exchange in combination with mass spectrometry (HXMS). In addition, gas phase fragmentation behaviors of deuterated peptides are investigated, with the purpose of increasing resolution of the HXMS. In the area of single protein dynamics, two protein systems are studied. Studies on the cytochrome c2 from Rhodobacter capsulatus indicate its domain stability to be similar to that of the horse heart cytochrome c. Further comparison of the exchange kinetics of the cytochrome c2 in its reduced and oxidized state reveals that the so-called hinge region is destabilized upon oxidation. We also applied a similar approach to investigate the conformational changes of photoactive yellow protein when it is transiently converted from the resting state to the signaling state. The central β-sheet of the protein is shown to be destabilized upon photoisomerization of the double bond in the chromophore. Another equally important question when it comes to understanding how proteins work is the interactions between proteins. To this end, two protein complexes are subjected to studies by solution phase hydrogen deuterium exchange and mass spectrometry. In the case of LexA/RecA interaction, both proteins show decreases in their extents of exchange upon complex formation. The potential binding site in LexA was further mapped to the same region that the protein uses to cleave itself upon interacting with RecA. In the sHSP/MDH system, hydrogen/deuterium exchange experiments revealed regions within sHSP-bound MDH that were significantly protected against exchange under heat denaturing condition, indicative of a partially unfolded state. Hydrogen/deuterium exchange therefore provides a way of probing low resolution protein structure within protein complexes that have a high level of heterogeneity. Finally, the feasibility of increasing resolution of HXMS by gas phase peptide fragmentation is investigated by using a peptide with three prolines near the C-terminus. Our data show that deuterium migration indeed occurs during the collision activated dissociation process. Caution is required when interpreting the MS/MS spectra as a way of pinpointing the exact deuterium distribution within peptides.
Type:
text; Electronic Dissertation
Keywords:
Mass Spectrometry; Protein dynamics; protein/protein interaction; non-covalent protein complex
Degree Name:
PhD
Degree Level:
doctoral
Degree Program:
Chemistry; Graduate College
Degree Grantor:
University of Arizona
Advisor:
Wysocki, Vicki H.
Committee Chair:
Wysocki, Vicki H.

Full metadata record

DC FieldValue Language
dc.language.isoENen_US
dc.titleUnraveling Macro-Molecular Machinery by Mass Spectrometry: from Single Proteins to Non-Covalent Protein Complexesen_US
dc.creatorCheng, Guilongen_US
dc.contributor.authorCheng, Guilongen_US
dc.date.issued2007en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractPresented in this dissertation are studies of protein dynamics and protein/protein interactions using solution phase hydrogen/deuterium exchange in combination with mass spectrometry (HXMS). In addition, gas phase fragmentation behaviors of deuterated peptides are investigated, with the purpose of increasing resolution of the HXMS. In the area of single protein dynamics, two protein systems are studied. Studies on the cytochrome c2 from Rhodobacter capsulatus indicate its domain stability to be similar to that of the horse heart cytochrome c. Further comparison of the exchange kinetics of the cytochrome c2 in its reduced and oxidized state reveals that the so-called hinge region is destabilized upon oxidation. We also applied a similar approach to investigate the conformational changes of photoactive yellow protein when it is transiently converted from the resting state to the signaling state. The central β-sheet of the protein is shown to be destabilized upon photoisomerization of the double bond in the chromophore. Another equally important question when it comes to understanding how proteins work is the interactions between proteins. To this end, two protein complexes are subjected to studies by solution phase hydrogen deuterium exchange and mass spectrometry. In the case of LexA/RecA interaction, both proteins show decreases in their extents of exchange upon complex formation. The potential binding site in LexA was further mapped to the same region that the protein uses to cleave itself upon interacting with RecA. In the sHSP/MDH system, hydrogen/deuterium exchange experiments revealed regions within sHSP-bound MDH that were significantly protected against exchange under heat denaturing condition, indicative of a partially unfolded state. Hydrogen/deuterium exchange therefore provides a way of probing low resolution protein structure within protein complexes that have a high level of heterogeneity. Finally, the feasibility of increasing resolution of HXMS by gas phase peptide fragmentation is investigated by using a peptide with three prolines near the C-terminus. Our data show that deuterium migration indeed occurs during the collision activated dissociation process. Caution is required when interpreting the MS/MS spectra as a way of pinpointing the exact deuterium distribution within peptides.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectMass Spectrometryen_US
dc.subjectProtein dynamicsen_US
dc.subjectprotein/protein interactionen_US
dc.subjectnon-covalent protein complexen_US
thesis.degree.namePhDen_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineChemistryen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorWysocki, Vicki H.en_US
dc.contributor.chairWysocki, Vicki H.en_US
dc.contributor.committeememberVierling, Elizabethen_US
dc.contributor.committeememberSaavedra, S. Scotten_US
dc.contributor.committeememberAspinwall, Craig A.en_US
dc.contributor.committeememberCusanovich, Michael A.en_US
dc.identifier.proquest2099en_US
dc.identifier.oclc659747208en_US
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