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

dc.contributor.authorLin, Bettyen
dc.contributor.supervisorWu, Gangen
dc.contributor.supervisorSmith, Stevenen'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.relation.ispartofseriesCanadian thesesen
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dc.subjectSolid-State NMRen
dc.subject17O Protein NMRen
dc.subject17O Labelingen
dc.titleAmino Acid-Type Specific Incorporation of Stable 17O Isotopes into Yeast Ubiquitinen
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