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Title: Biophysical studies of cell cycle regulatory proteins
Author: Hutton, Richard David
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2006
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Studies performed on the multi-domain cell cycle regulatory protein Cdk2 showed that the protein unfolds in a 3-state manner. The intermediate converts to the native state on a very slow timescale. Mutation analysis shows that the protein can accommodate mutations at lower energetic cost than small single domain proteins, presumably by rearrangement of the structure. Kinetic studies show that the β-sheet N-terminal lobe unfolds rapidly followed by the slower unfolding of the α-helical C-lobe. Analysis of the refolding kinetics was hampered by the presence of a cis praline in the structure. However, mutation of this praline to alanine enabled a fast refolding phase to be detected. The 226 residue protein gankyrin is composed of seven ankyrin repeats that stack linearly to form an elongated, predominately α-helical structure. The protein unfolds in a reversible and two-state manner at equilibrium with a free energy of unfolding in water of 10.8 kcal mol-1. The chevron plot is described by at least four different regimes, giving rise to different types of non-linearity. At the very lowest denaturant concentrations the rate constant increases with increasing denaturant concentrations, suggestive of an off-pathway intermediate. At slightly higher denaturant concentrations there is rollover in the refolding limb, indicating the formation of another folding intermediate. In the early part of the unfolding limb of the chevron plot, upward curvature is observed, rationalised as parallel pathways. In the later part of the unfolding limb, downward curvature is observed and this behaviour was fitted according to a sequential transition states model. The order of structure formation in the folding of gankyrin was probed using protein engineering techniques.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available