ENZYME-FREE UBIQUITIN LIGATION. FROM NATIVE CHEMICAL LIGATION AND SPIN LABELING TO DIMERIZATION BY INTER-UBIQUITIN MIMICKING LINKAGE AND PH-DEPENDENT CONFORMATIONAL SWITCH

dc.contributor.advisorFushman, Daviden_US
dc.contributor.authorHerrera-Guzman, Karinaen_US
dc.contributor.departmentChemistryen_US
dc.contributor.publisherDigital Repository at the University of Marylanden_US
dc.contributor.publisherUniversity of Maryland (College Park, Md.)en_US
dc.date.accessioned2016-09-03T05:33:48Z
dc.date.available2016-09-03T05:33:48Z
dc.date.issued2016en_US
dc.description.abstractThe delicate balance between the production and disposal of proteins is vital for the changes required in the cell to respond to given stimulus. Ubiquitination is a protein modification with a range of signaling outcomes when ubiquitin is attached to a protein through a highly ordered enzymatic cascade process. Understanding ubiquitination is a growing field and nowadays the application of chemical reactions allows the isolation of quantitative materials for structural studies. Therefore, in this dissertation it is described some of these suitable chemical methodologies to produce an isopeptide bond toward the polymerization of ubiquitin bypassing the enzymatic control with the purpose of showing if these chemical modifications have a direct impact on the structure of ubiquitin. First, the possibility of incorporating non-natural lysine analogs known as mercaptolysines into the polypeptide chain of Ubiquitin was explored when they were attached to ubiquitin by native chemical ligation at its C terminus. The sulfhydryl group was used for the attachment of a paramagnetic label to map the surface of ubiquitin. Second, the condensation catalyzed by silver nitrate was used for the dimer assembly. In particular, the main focus was on examining whether orthogonal protection and deprotection of each monomer have an impact on the reaction yield, since the synthetic strategy has been previously attempted successfully. Third, the formation of ubiquitin dimers was approached by building an inter-ubiquitin linkage mimicking the isopeptide bond with two approaches, the classic disulfide exchange as well as the thiol-ene click reaction by thermal initiation in aqueous conditions. After assembling the dimeric units, they were studied by Nuclear Magnetic Resonance, in order to establish a conformational state profile which depends on the pH conditions. The latter is a very important concept since some ligands have a preferred affinity when the protein-protein hydrophobic patches are in close proximity.en_US
dc.identifierhttps://doi.org/10.13016/M2FV3F
dc.identifier.urihttp://hdl.handle.net/1903/18540
dc.language.isoenen_US
dc.subject.pqcontrolledBiochemistryen_US
dc.subject.pqcontrolledOrganic chemistryen_US
dc.subject.pquncontrolleddimerizationen_US
dc.subject.pquncontrolledisopeptide mimicsen_US
dc.subject.pquncontrollednative chemical ligationen_US
dc.subject.pquncontrolledubiquitinen_US
dc.titleENZYME-FREE UBIQUITIN LIGATION. FROM NATIVE CHEMICAL LIGATION AND SPIN LABELING TO DIMERIZATION BY INTER-UBIQUITIN MIMICKING LINKAGE AND PH-DEPENDENT CONFORMATIONAL SWITCHen_US
dc.typeDissertationen_US

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