Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.659723
Title: Synthesis and conformational studies of a protein
Author: Muir, Thomas W.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1992
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Abstract:
An investigation into the conformational stability of the small globular protein ubiquitin (Ub) is described. Solid phase peptide synthesis has been successfully applied to the construction of both mammalian ubiquitin, as well as to a ubiquitin analogue (Ubdes-Core) lacking the protein's pronounced hydrophobic core. In each case the synthetic protein was purified to homogeneity using a combination of gel filtration, dialysis and ion exchange chromatography. Synthetic ubiquitin was found to be fully active in a ubiquitin specific in vitro protein conjugation assay, whereas Ubdes-Core was found to be completely inactive. The structure of these two proteins has been studied in some detail. Synthetic ubiquitin possesses an identical crystal and NMR derived solution structure to its natural counterpart. Removal of the hydrophobic core from ubiquitin results in a substantial loss in conformational stability (3.7 kcal mol-1) and consequently Ubdes-Core is unable to adopt the ubiquitin native fold. This leads to the conclusions that hydrogen bonding contributions are not in themselves sufficient to stabilize the native conformation of ubiquitin. The preparation of a 128 residue precursor protein comprising ubiquitin fused to a C-terminal extension protein (CE-52) is presented along with the CEP52 fragment alone. The compounds were prepared by solid phase peptide synthesis and purified using a combination of gel filtration, ion exchange and HPLC. Two-dimensional NMR analysis of the UbCEP52 precursor revealed, significantly, that it possesses no well defined tertiary structure. However, the protein was found to be processed in vitro to mature ubiquitin and CEP52. A possible novel function of ubiquitin in unfolded C-terminally fused conujugates is suggested.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.659723  DOI: Not available
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