Expanding the Role of Gas-Phase Methods in Structural Biology: Characterization of Protein Quaternary Structure and Dynamics by Tandem Mass Spectrometry and Ion Mobility

Persistent Link:
http://hdl.handle.net/10150/238912
Title:
Expanding the Role of Gas-Phase Methods in Structural Biology: Characterization of Protein Quaternary Structure and Dynamics by Tandem Mass Spectrometry and Ion Mobility
Author:
Blackwell, Anne
Issue Date:
2012
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:
This dissertation presents efforts to expand the role of mass spectrometry (MS) in structural biology. Determination of quaternary structure of a protein complex has been hindered by limited fragmentation from collision-induced dissociation (CID). As an alternative, surface-induced dissociation (SID) was implemented for a quadrupole - time-of-flight instrument in the Wysocki laboratory. This research tested the hypothesis that SID should produce fragmentation reflective of subunit organization. Furthermore, ion mobility (IM) was used to prove the direct relationship between precursor conformation and observed dissociation patterns, and the relationship between activation and product ion conformation. The structure and dynamics of a dimeric small heat shock protein (sHSP) with no solved structure was investigated. The importance of N- and C-terminal domains for dimerization was determined, and the dimers were shown to exchange subunits. From exchange kinetics it is proposed that subunit exchange is unrelated to heat shock activity. SID was used to elucidate the subunit architecture of heterogeneous protein assemblies, including one previously solved protein structure and two formerly uncharacterized proteins. The heterohexamer toyocamycin nitrile hydratase dissociated into trimers, revealing the hexamer to be a dimer of trimers. The bacterial ribonuclease toxin:antitoxin tetramer was shown to have an antitoxin dimer at its core, with flanking individual toxin subunits. The examples presented here are the first clear proof that SID results can clearly indicate the substructure of a protein assembly.IM was used to study the conformation of precursor and product ions. A greater understanding of the relationship between precursor conformation and observed dissociation patterns was developed. Different charge states of a dodecameric sHSP were found to have significantly different conformations, which were directly reflected in SID spectra. IM comparison of CID and SID product ions showed that the same charge state of a product ion from either method has the same CCS. This suggests the product ion conformation is dependent upon ion charge state, and independent of activation method and collision energy. The cause and effect relationship between precursor conformation and MS/MS patterns, and activation and product ion conformation were clearly illustrated. Together, this body of research expands the role of MS for structural biology.
Type:
text; Electronic Dissertation
Keywords:
non-covalent protein complexes; quaternary structure; surface-induced dissociation; Chemistry; ion mobility; mass spectrometry
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Chemistry
Degree Grantor:
University of Arizona
Advisor:
Wysocki, Vicki H.

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleExpanding the Role of Gas-Phase Methods in Structural Biology: Characterization of Protein Quaternary Structure and Dynamics by Tandem Mass Spectrometry and Ion Mobilityen_US
dc.creatorBlackwell, Anneen_US
dc.contributor.authorBlackwell, Anneen_US
dc.date.issued2012-
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.abstractThis dissertation presents efforts to expand the role of mass spectrometry (MS) in structural biology. Determination of quaternary structure of a protein complex has been hindered by limited fragmentation from collision-induced dissociation (CID). As an alternative, surface-induced dissociation (SID) was implemented for a quadrupole - time-of-flight instrument in the Wysocki laboratory. This research tested the hypothesis that SID should produce fragmentation reflective of subunit organization. Furthermore, ion mobility (IM) was used to prove the direct relationship between precursor conformation and observed dissociation patterns, and the relationship between activation and product ion conformation. The structure and dynamics of a dimeric small heat shock protein (sHSP) with no solved structure was investigated. The importance of N- and C-terminal domains for dimerization was determined, and the dimers were shown to exchange subunits. From exchange kinetics it is proposed that subunit exchange is unrelated to heat shock activity. SID was used to elucidate the subunit architecture of heterogeneous protein assemblies, including one previously solved protein structure and two formerly uncharacterized proteins. The heterohexamer toyocamycin nitrile hydratase dissociated into trimers, revealing the hexamer to be a dimer of trimers. The bacterial ribonuclease toxin:antitoxin tetramer was shown to have an antitoxin dimer at its core, with flanking individual toxin subunits. The examples presented here are the first clear proof that SID results can clearly indicate the substructure of a protein assembly.IM was used to study the conformation of precursor and product ions. A greater understanding of the relationship between precursor conformation and observed dissociation patterns was developed. Different charge states of a dodecameric sHSP were found to have significantly different conformations, which were directly reflected in SID spectra. IM comparison of CID and SID product ions showed that the same charge state of a product ion from either method has the same CCS. This suggests the product ion conformation is dependent upon ion charge state, and independent of activation method and collision energy. The cause and effect relationship between precursor conformation and MS/MS patterns, and activation and product ion conformation were clearly illustrated. Together, this body of research expands the role of MS for structural biology.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectnon-covalent protein complexesen_US
dc.subjectquaternary structureen_US
dc.subjectsurface-induced dissociationen_US
dc.subjectChemistryen_US
dc.subjection mobilityen_US
dc.subjectmass spectrometryen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineChemistryen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorWysocki, Vicki H.en_US
dc.contributor.committeememberAspinwall, Craig A.en_US
dc.contributor.committeememberBandarian, Vaheen_US
dc.contributor.committeememberPemberton, Jeanne E.en_US
dc.contributor.committeememberWysocki, Vicki H.en_US
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