Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.550343
Title: Characterising the unfolded state of Im7 in non-denaturing conditions
Author: Pashley, Clare Louise
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2011
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Abstract:
An understanding of the structural ensemble of the species present at the commencement of protein folding may help to elucidate the role of the primary amino acid sequence in narrowing the search for the native conformation during protein folding. Characterisation of this species is often difficult since the folded state is the predominantly populated species at equilibrium. In addition to this, the study of disordered proteins suffers from a lack of restraints, since the protein can adopt many different conformations in solution. However, advances in computational methods now make it possible to build an ensemble model of the unfolded state using experimentally measured parameters. Previous work has shown the 87-residue, 4-helix protein, Im7, folds with a three-state mechanism via an on-pathway intermediate. While the transition states and populated intermediate on the folding pathway have been characterised in atomistic detail, knowledge of the unfolded ensemble under the same ambient conditions remained sparse. Although the urea denatured state of Im7 has been characterised, single molecule experiments revealed that this species of Im7 becomes compact upon dilution of the denaturant. These observations suggest that the structural ensemble of the unfolded state of Im7 is different in the absence of urea. In this thesis, destabilising amino acid substitutions were introduced into the sequence of Im7, such that the unfolded state is predominantly populated at equilibrium in the absence of denaturant. Results from far and near-UV CD, fluorescence, urea titration and heteronuclear NMR experiments reveal that three amino acid substitutions (L18A L19A L37A) are sufficient to prevent Im7 folding. Using this variant the unfolded state of Im7 under ambient conditions was then shown to be more compact, and to have increased tendency towards sampling the a-region of Φ/Ψ space compared with the protein in 6 M urea. Measurements of 15N-transverse relaxation rates revealed that sequences corresponding to helical regions in the native protein are conformationally restricted within the unfolded ensemble as a consequence of local hydrophobic clustering without substantial helix formation. By creating hydrophobic deletion mutants and measuring paramagnetic . relaxation effects, possible transient interactions between regions of the sequence relating to the native helix IV and the N-terminal region of the protein were identified, which may restrict conformational sampling within the ensemble. An atomistic model of the unfolded ensemble is then presented to create the first atomistic impression of the unfolded state of Im7. This provides a powerful tool for designing future experiments for refinement of the presented model, and for probing the effect of unfolded state interactions on the folding pathway of Im7.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.550343  DOI: Not available
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