Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.599147
Title: Folding studies on an immunoglobulin domain using chemical and mechanical methods
Author: Fowler, S. B.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2002
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Abstract:
Part I of this thesis focuses on the folding and stability of TI 127, the 27th Ig domain from the I-band of human cardiac titin, using chemical denaturants. The first comprehensive protein engineering analysis of the transition of an immunoglobulin (Ig) I-set domain is presented. Preliminary studies reveal that TI 127 folds via a kinetic intermediate. The transition state shifts to a less native-like state upon mutation, an indication of strain in the hydrophobic core. These two effects make a standard analysis impossible. However, the data can be used to identify critical residues involved in the transition state for folding. The transition state structure is compared with those of three other Ig-like domains from different evolutionary superfamilies and with little sequence homology. Results show that for all four domains, folding is driven by a ring of interacting residues in the common structural core. This indicates that the folding pathway is driven by thermodynamic features of the fold rather than evolutionary constraints. The I-band of titin is composed principally of Ig domains arranged in tandem and is subjected to applied force in fulfilling its function in vivo. These domains must resist force and also be able to unfold and recover. Atomic force microscopy (AFM) is a relatively new tool for studying protein unfolding in the presence of applied force. In part II a detailed analysis of the unfolding of TI 127 is presented and compared with results form Part I. Under applied force, TI 127 unfolds via a stable intermediate and a model for its structure is proposed. Protein engineering analysis shows that the loss of the same set of interactions in the structure initiates unfolding using both denaturants and AFM. If unfolding under applied force reflects events in vivo, then this indicates that kinetic information from studies using denaturants may be directly applicable to in vivo behaviour.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.599147  DOI: Not available
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