The protein ubiquitin has been used as a model for folding studies with the synthesis of ubiquitin analogues including both single and double substitutions. Chemical synthesis has allowed for the facile incorporation of both unnatural amino acids and a novel synthetic fluorinated amino acid. Purification of synthetic ubiquitin by a variety of protocols has identified important steps in the folding pathway. We have investigated the placement of fluorine within the hydrophobic core of ubiquitin through the synthesis of three double substitutions at positions 43&67, 50&67, 56&67 and one single substitution at position 67 using (2S,4S)-5-fluoroleucine to replace leucine. Methyl transfer across the hydrophobic core has been considered using the double substitution analogue [3-Norleucine,43-norvaline]ubiquitin involving a modification in the distribution of alkyl groups. The importance of the chirality of the single histidine on the final strand of β-sheet has been examined in the synthesis of the analogue [68-DHistidine]ubiquitin. The contribution of secondary structural features in protein folding has been investigated by studying peptide fragments corresponding to the N and C-terminal halves of ubiquitin and three overlapping peptides on the final strand of β-sheet. The complementation of the N and C-terminal halves to regenerate the native fold has also been studied.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:663777 |
| Date | January 1995 |
| Creators | Wilken, Jill |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/13236 |
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