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Title: The role of the N-terminal beta-hairpin in the folding of ubiquitin
Author: Chen, P. H. R.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 1998
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It has been found in previous studies that the N-terminal sequence, 1-17, of ubiquitin has a strong β-hairpin forming propensity so that a synthetic peptide corresponding to this sequence, U(1-17), can populate a β-hairpin structure even in aqueous solution. Mutual stabilisation between the N-terminal β-hairpin and the following α-helix has also been found in comparative studies of several peptides which contain these two segments individually, U(1-17), U(21-35), or together, U(1-35). The data suggest the possible importance of the N-terminal β-hairpin in the folding of ubiquintin. In addition, they suggest that this peptide could provide an invaluable experimental system for studying aspects of β-sheet folding. In this project a protein engineering strategy has been used to explore the factors that determine the stability and structural specificity of the hairpin. Several mutations were made in the turn region (T7V, T9A, T9D, T9N, desG10) and on one strand (F4A, F4W) of the hairpin. In order to explore the effects of mutations on both the whole protein and the peptide segments, two cleavage mutations were introduced: at the end of the β-hairpin (E18D) and at the end of the α-helix (D39C). Unfortunately, due to the physical properties of the cleaved peptides, the yield of the chemical cleavage at the E18D mutation site was not satisfactory, hence most of the peptide results were ultimately obtained from studies of synthetic peptides. The yield of the cleavage of D39C was even worse, probably because of steric hindrance from the preceding residues, Pro-37 and 38; therefore no studies of the longer peptide (1-38) could be undertaken. The results show that Gly-10 in the bulge of the β-turn is very important to the stability of the hairpin. Deleting this residue unfolds the structure of the peptide desG10(1-16) in water. Remarkably, however, the native state of ubiquitin is barely destabilised by the same deletion. This may be due, at least in part, to the fact that Gly-10 appears also to be important in maintaining residual structure of ubiquitin in the denatured state. In refolding experiments, ubiquitin was found to show biphasic kinetics at low pH. The desG10 mutation affected mainly the faster process, which may support the prediction that the N-terminal hairpin is involved in the early stages of folding of this protein.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available