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Production, Characterization and Structure Determination of the C-terminal Domain of Stt3p: the Catalytic Subunit of Yeast Oligosaccharyl Transferase


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dc.contributor.advisorMohanty, Smita
dc.contributor.authorHuang, Chengdong
dc.date.accessioned2010-08-03T13:32:03Z
dc.date.available2010-08-03T13:32:03Z
dc.date.issued2010-08-03T13:32:03Z
dc.identifier.urihttp://hdl.handle.net/10415/2277
dc.description.abstractN-glycosylation, the most ubiquitous protein modification in eukaryotes, is catalyzed by the enzyme complex Oligosaccharyl Transferase (OT). This protein co-translational modification has been implicated in a multitude of cellular processes, and defects in the N-glycosylation cause a group of inherited human disorders known as Congenital Disorders of Glycosylation (CDG), while complete loss of N-linked glycosylation is lethal to all eukaryotic organisms. In the key reaction of N-glycosylation, OT transfers preassembled oligosaccharide moieties from lipid-linked donors onto the asparagine residues in a consensus sequence of Asn-Xaa-Thr/Ser (where Xaa ≠ proline) on nascent polypeptides. For eukaryotes, OT is a remarkably complex multisubunit enzyme that, in the case of the yeast Saccharomyces cerevisiae, contains nine nonidentical integral membrane protein subunits, among which Wbp1, Swp1, Ost1, Ost2, and Stt3 proteins are essential for the viability of cells. Although the detailed enzymatic reaction mechanism and the roles of the other subunits are not yet fully understood, a multitude of experimental evidences show that the C-terminal domain of Stt3p is the catalytic domain of the OT complex. My doctoral dissertation is primarily focused on the following three parts: (1) production, (2) biophysical characterization and (3) 3D structure determination of the C-terminal Stt3p by high-resolution solution NMR. The C-terminal domain of Stt3p was expressed at 60~70 mg/L in E. coli and purified by a robust but novel method which has been developed by our lab, “SDS Elution”. Circular Dichroism (CD) and NMR spectra indicate that the C-terminal Stt3p is highly helical and has a stable tertiary structure in SDS micelles. In addition, the comparative analysis of the CD, fluorescence and NMR data of the mutant and the wild-type protein revealed that the replacement of the key residue Asp518, which is located within the W516WDYG520 signature motif, led to a distinct tertiary structure, even though both proteins have similar overall secondary structures. This observation strongly suggests that Asp518, which was previously proposed to primarily function as a catalytic residue, also plays a critical structural role. Moreover, the activity of the protein was confirmed by saturation transfer difference (STD) and NMR titration studies. For NMR structure determination, approximately 93% of the backbone resonances and most of the side-chain resonances have been assigned. To determine the atomic-resolution solution structure of the C-terminal domain of Stt3p, so far the largest α-helical integral membrane protein whose structure is to be determined by NMR, a combination of various constraints have used, including NOEs from {15N, 13C}-double-labeled, partially deuterated (50%) triple-labeled, uniformly {2H, 13C, 15N}-triple-labeled, and ILV methyl protonated otherwise uniformly {2H, 13C, 15N}-triple-labeled sample, together with backbone dihedral angles from chemical shift analysis (TALOS+), residual dipolar couplings (RDCs) and paramagnetic relaxation enhancement (PRE) measurements from 15 nitroxide labeled samples. At the end, we were able to determine the 3D structure of the C-terminal domain of Stt3p. To date, this is the first high-resolution structure of the catalytic domain of the eukaryotic OT complex. Considering the high sequence homology among eukaryotic Stt3ps, we hope our results can provide a significant step toward the structural understanding of the mechanisms of the N-glycosylation in eukaryotes.en
dc.rightsEMBARGO_NOT_AUBURNen
dc.subjectChemistry and Biochemistryen
dc.titleProduction, Characterization and Structure Determination of the C-terminal Domain of Stt3p: the Catalytic Subunit of Yeast Oligosaccharyl Transferaseen
dc.typedissertationen
dc.embargo.lengthMONTHS_WITHHELD:6en_US
dc.embargo.statusEMBARGOEDen_US
dc.embargo.enddate2011-02-03en_US

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