PROBING GAS-PHASE PEPTIDE STRUCTURE AND PROTEIN-PROTEIN INTERACTIONS USING MASS SPECTROMETRIC TECHNIQUES

Persistent Link:
http://hdl.handle.net/10150/194317
Title:
PROBING GAS-PHASE PEPTIDE STRUCTURE AND PROTEIN-PROTEIN INTERACTIONS USING MASS SPECTROMETRIC TECHNIQUES
Author:
Perkins, Brittany
Issue Date:
2009
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 on the gas-phase structural features of peptides and peptide fragment ions using mass spectrometry (MS), hydrogen/deuterium (H/D) exchange, infrared multiphoton dissociation (IRMPD) spectroscopy, and computational modeling. Additional studies are presented on the mechanism of hydrogen/deuterium exchange using a model amino acid system. The application of chemical cross-linking to investigate the interaction between two proteins, LexA and RecA, is also presented. Gas-phase structural features can be probed using a number of techniques, and several of the studies presented in this dissertation involve the use of gas-phase H/D exchange. Although the basic mechanism for exchange has been determined, the factors that affect the rate and extent of exchange are not well understood. A computational modeling study of the exchange behavior of asparagine and its methyl ester demonstrated that exchange will occur preferentially at sites of more similar basicity. The distinctive exchange behavior of a model histidine-containing pentapeptide, HAAAA, prompted further studies into the structural features that result in five fast exchanging hydrogens and one slower exchange. Peptide analogues were used to identify the sites of exchange, and IRMPD spectroscopy combined with computational modeling indicated that exchange may occur because interaction with water at those sites results in lower energy structures compared to the other sites. Structural studies were also performed to determine whether the b₂⁺ ion from HAAAA is an oxazolone or diketopiperazine. Although the IRMPD spectrum matched that of a diketopiperazine, H/D exchange and fragmentation studies revealed the presence of both a diketopiperazine and oxazolone structure. Protein-protein interactions perform a vital role in regulating cellular processes. Despite extensive mutational analysis, the binding interaction between LexA and RecA, two proteins involved in the SOS response, is unclear. Chemical cross-linking experiments were undertaken to help target future mutational studies, and these studies identified two possible interactions. The first potential binding interaction is located in the cleft of RecA, and the second interaction may be caused by a LexA dimer binding across the RecA helical groove. The presence of two different binding interactions suggests that LexA may have redundant binding modes for RecA interaction.
Type:
text; Electronic Dissertation
Keywords:
chemical cross-linking; gas-phase structure; H/D exchange; mass spectrometry; protein-protein interactions
Degree Name:
Ph.D.
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.titlePROBING GAS-PHASE PEPTIDE STRUCTURE AND PROTEIN-PROTEIN INTERACTIONS USING MASS SPECTROMETRIC TECHNIQUESen_US
dc.creatorPerkins, Brittanyen_US
dc.contributor.authorPerkins, Brittanyen_US
dc.date.issued2009en_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 on the gas-phase structural features of peptides and peptide fragment ions using mass spectrometry (MS), hydrogen/deuterium (H/D) exchange, infrared multiphoton dissociation (IRMPD) spectroscopy, and computational modeling. Additional studies are presented on the mechanism of hydrogen/deuterium exchange using a model amino acid system. The application of chemical cross-linking to investigate the interaction between two proteins, LexA and RecA, is also presented. Gas-phase structural features can be probed using a number of techniques, and several of the studies presented in this dissertation involve the use of gas-phase H/D exchange. Although the basic mechanism for exchange has been determined, the factors that affect the rate and extent of exchange are not well understood. A computational modeling study of the exchange behavior of asparagine and its methyl ester demonstrated that exchange will occur preferentially at sites of more similar basicity. The distinctive exchange behavior of a model histidine-containing pentapeptide, HAAAA, prompted further studies into the structural features that result in five fast exchanging hydrogens and one slower exchange. Peptide analogues were used to identify the sites of exchange, and IRMPD spectroscopy combined with computational modeling indicated that exchange may occur because interaction with water at those sites results in lower energy structures compared to the other sites. Structural studies were also performed to determine whether the b₂⁺ ion from HAAAA is an oxazolone or diketopiperazine. Although the IRMPD spectrum matched that of a diketopiperazine, H/D exchange and fragmentation studies revealed the presence of both a diketopiperazine and oxazolone structure. Protein-protein interactions perform a vital role in regulating cellular processes. Despite extensive mutational analysis, the binding interaction between LexA and RecA, two proteins involved in the SOS response, is unclear. Chemical cross-linking experiments were undertaken to help target future mutational studies, and these studies identified two possible interactions. The first potential binding interaction is located in the cleft of RecA, and the second interaction may be caused by a LexA dimer binding across the RecA helical groove. The presence of two different binding interactions suggests that LexA may have redundant binding modes for RecA interaction.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectchemical cross-linkingen_US
dc.subjectgas-phase structureen_US
dc.subjectH/D exchangeen_US
dc.subjectmass spectrometryen_US
dc.subjectprotein-protein interactionsen_US
thesis.degree.namePh.D.en_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.committeememberAspinwall, Craig A.en_US
dc.contributor.committeememberLichtenberger, Dennisen_US
dc.contributor.committeememberLittle, Johnen_US
dc.identifier.proquest10580en_US
dc.identifier.oclc659752324en_US
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