Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.596604
Title: High resolution studies of protein folding and dynamics
Author: Best, R.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2003
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Abstract:
This thesis presents several approaches to the study of the folding and dynamics of proteins, with an emphasis on examples form immunoglobulin-like fold. The first part describes the application of atomic force microscopy to studying the mechanical unfolding of proteins. Most studies to date have used methods such as chemical or thermal denaturation, thus force represents a new type of “denaturant” which adds to the description of the folding energy landscape. Furthermore it is a single-molecule method with the potential to resolve parallel folding pathways, for example. The technique has been used to study the mechanical unfolding of the RNase barnase which has already been extensively characterised by more conventional methods. Barnase is shown to be folded and stable in the protein construct used, yet it unfolds at very low forces compared to proteins with mechanical function (such as titin 127). Phi-value analysis by protein engineering has allowed the detailed characterization of folding pathways by bulk methods. A method is presented and justified which circumvents the large uncertainties in unfolding rates obtained from kinetic fits to AFM data. This has been applied to the domain titin 127, a protein from muscle, showing that the protein unfolds through an intermediate and with a very native-like transition state under force, contrasting with previous bulk studies. The second part of the work concerns the comparison of TNfn3 and FNfn10, belonging to the fibronectin type III superfamily and, like 127, members of the immunoglobulin-like fold. These two domains have very similar structure, yet present a very different response to mutation, which has led to the suggestion that FNfn10 has greater structural “plasticity”. The dynamics of these domains are addressed by means of equilibrium hydrogen exchange measurements, which support the notion that the periphery of FNfn10 is more flexible. The core dynamics have been investigated using recently developed NMR side-chain dynamics experiments, and the results compared to molecular dynamics simulations.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.596604  DOI: Not available
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