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Development of Microfluidic Devices for Studying Protein Conformational Dynamics via HDX/TRESI-MS

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dc.contributor.advisorWilson, Derek J.
dc.creatorRob, Tamanna
dc.date.accessioned2014-07-31T14:59:18Z
dc.date.available2014-07-31T14:59:18Z
dc.date.copyright2013-06-21
dc.date.issued2014-07-28
dc.date.updated2014-07-28T16:16:46Z
dc.degree.disciplineChemistry
dc.degree.levelDoctoral
dc.degree.namePhD - Doctor of Philosophy
dc.description.abstractStudies of protein conformational dynamics can lead to a deeper understanding of biological functions of proteins, protein/protein interactions, catalysis and allostery. Most of the conformers involved in biological pathways are short-lived. These conformers represent challenging analytical targets since they are weakly populated at equilibrium and demand ultra-rapid online analytical techniques. To address this demand, the development of new tools that will offer enhanced time and spatial resolution in measurements is essential. Towards that end, we took advantage of microfluidics to fabricate custom polymeric devices that would allow direct mass spectrometric measurements of protein dynamics. This dissertation presents successful development of new tools and demonstrates their practical applications in studying dynamics of weakly structured regions of proteins on the millisecond time scale, which is unattainable by conventional biophysical techniques. This thesis introduces a simple, rapid, inexpensive and easily modifiable fabrication protocol which enables the integration of multiple functionalities on a single platform that accommodates complete ‘bottom up’ continuous/pulse labelling Hydrogen-deuterium exchange (HDX) work flow. These integrated devices can be directly interfaced with mass spectrometry as custom electrospray ionization (ESI) sources to conduct site specific mass spectrometric protein conformational analysis. The continuous HDX labelling device enables structural analysis of ubiquitin and distinguishes loop dynamics in cytochrome c. The effect of ligand binding in conformational dynamics in a molten globule protein DAHP synthase was also monitored via continuous HDX labelling. Another analogous device, capable of performing millisecond HDX pulse labelling, reveals allosteric hot spots in TEM-1 which is an antibiotic resistant β-lactamase. Complementary experiments with multiple antibiotics compare the allosteric responses of TEM-1 upon inhibitor or substrate binding. The results exhibit an exciting correlation between allosteric dynamic responses and functionally relevant mutations at the periphery of the protein. This strong correlation demonstrates that this device can be applied in predicting sites of probable mutations. This dissertation successfully introduces integrated microfluidic devices that represent a promising tool for studying dynamics of weakly structured proteins, intrinsically disordered proteins and allostery.en_US
dc.identifier.urihttp://hdl.handle.net/10315/27692
dc.language.isoenen_US
dc.rightsAuthor owns copyright, except where explicitly noted. Please contact the author directly with licensing requests.
dc.subjectChemistryen_US
dc.subjectAnalytical chemistryen_US
dc.subject.keywordsPMMAen_US
dc.subject.keywordsMass Spectrometry (MS)en_US
dc.subject.keywordsElectrospray Ionization (ESI)en_US
dc.subject.keywordsTime resolved ESI-MS (TRESI-MS)en_US
dc.subject.keywordsMicrofluidicsen_US
dc.subject.keywordsProtein dynamicsen_US
dc.subject.keywordsProtein conformational dynamicsen_US
dc.subject.keywordsTime resolved protein dynamics studiesen_US
dc.subject.keywordsMillisecond time scaleen_US
dc.subject.keywordsHydrogen-deuterium exchange (HDX)en_US
dc.subject.keywordsTEM-1en_US
dc.subject.keywordsDAHP synthaseen_US
dc.titleDevelopment of Microfluidic Devices for Studying Protein Conformational Dynamics via HDX/TRESI-MSen_US
dc.typeElectronic Thesis or Dissertation

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