Amino Acid-Type Specific Incorporation of Stable 17O Isotopes into Yeast Ubiquitin

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dc.contributor.authorLin, Bettyen
dc.contributor.departmentChemistryen
dc.contributor.supervisorWu, Gangen
dc.contributor.supervisorSmith, Stevenen
dc.date.accessioned2019-10-02T21:58:56Z
dc.date.available2019-10-02T21:58:56Z
dc.degree.grantorQueen's University at Kingstonen
dc.description.abstractStable isotopic labeling is an important part of structural biology by multidimensional NMR spectroscopy. Novel stable isotopic labeling methods have continuously been developed to enhance protein NMR spectroscopy. Protein structural determination based on 15N- and 13C-labelled proteins is already well established. However, oxygen, being one of the most abundant elements in biological molecules, has not yet been utilized in NMR studies of proteins. This is largely because of two factors. First, the only NMR-active oxygen isotope, 17O, has a very low natural abundance (0.037%). Second, 17O has a nuclear spin of 5/2 (known as quadrupolar), which usually results in broad 17O NMR signals even for small organic molecules. Despite the technical difficulties, recent studies have shown 17O NMR to be a valuable tool for studying biological molecules. In this thesis, we used yeast ubiquitin (8.6 kDa) as a model system to investigate general strategies of incorporating 17O-labelled amino acids into proteins. We have used an auxotrophic E.coli strain DL39 GlyA λDE3 (aspC- tyrB- ilvE- glyA- λDE3) to successfully synthesize yeast ubiquitin where glycine, phenylalanine, tyrosine, and alanine residues are selectively 17O-labelled. This is the first demonstration of 17O-labeling of a protein in both backbone groups and sidechain. Some preliminary solid-state 13C and 17O NMR results on these protein samples are also reported in this thesis. The synthetic methodology for 17O-labeling yeast ubiquitin reported in this thesis will be useful in future solid-state 17O NMR studies.en
dc.description.degreeM.Sc.en
dc.identifier.urihttp://hdl.handle.net/1974/26697
dc.language.isoengen
dc.relation.ispartofseriesCanadian thesesen
dc.rightsCC0 1.0 Universalen
dc.rightsQueen's University's Thesis/Dissertation Non-Exclusive License for Deposit to QSpace and Library and Archives Canadaen
dc.rightsProQuest PhD and Master's Theses International Dissemination Agreementen
dc.rightsIntellectual Property Guidelines at Queen's Universityen
dc.rightsCopying and Preserving Your Thesisen
dc.rightsThis publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.en
dc.rights.urihttp://creativecommons.org/publicdomain/zero/1.0/
dc.subject17Oen
dc.subjectNMRen
dc.subjectSolid-State NMRen
dc.subject17O Protein NMRen
dc.subject17O Labelingen
dc.titleAmino Acid-Type Specific Incorporation of Stable 17O Isotopes into Yeast Ubiquitinen
dc.typethesisen
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