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Title: Folding intermediates of wild-type and mutants of barnase
Author: Dalby, P. A.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 1998
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The folding of Bacillus Amyloliquefaciens RNase (barnase) is known to proceed via at least one intermediate before passing over the rate determining transition state to the native state. Here, I have used kinetic and equilibrium methods to study the co-operative nature of the intermediate formation, and also the movement of the intermediate and transition state on the folding energy landscape. Using similar methods, I have studied the progress of the formation of a network of secondary and tertiary interactions, as the protein folds, in order to determine whether the intermediate is on- or off-pathway. Finally, using the same kinetic and equilibrium methods, I have correlated changes in the stability of the intermediate to previously observed changes in the hydrogen exchange mechanism at equilibrium, then subsequently derived a simple kinetic scheme to explain these data. These results show that the intermediate is formed in a co-operative manner, with a first-order transition from the denatured state, although for some mutants, a second intermediate becomes populated close to the melting temperatures of the native proteins. Furthermore, mutational studies show that both the intermediate and transition state ensembles undergo structural redistribution on the energy landscape, as the temperature is varied. Their average structures do not appear to move on the energy landscape as the temperature is varied. However, the major α-helix of the transition state moves closer to the native state as the temperature is increased, in agreement with the Hammond effect observed previously at 7.25 M urea. These results also show that the intermediate contains only native interactions, which consolidate on going from the intermediate, to the transition state, and finally to the native state. Hence, the intermediate is on the folding pathway, and does not have to unfold again in order to reach the native state.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available