Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.639965
Title: A site-directed spin labelling study of the human alpha-lactalbumin molten globule
Author: Young, Matthew Alexander
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2013
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Abstract:
The human α-lactalbumin (α-LA) molten globule formed at low pH is a model for the study of protein folding intermediates. The molten globule lacks native-like side-chain interactions, resulting in a fluctuating ensemble of tertiary structures, characterisation of which has been precluded by severe line-broadening in NMR spectra and a lack of long-range NOEs. Paramagnetic relaxation enhancements (PREs) have been measured in a variant of α-LA in which all native cysteines have been mutated to alanine (all-Ala α-LA). Cysteine residues have been mutated into regions of interest and spin labelled with MTSL. These measurements have confirmed that all-Ala α-LA forms a compact molten globule. Transient, long-range interactions that are stabilising the compact fold have also been identified using PREs measured in urea-denatured states. This has identified several interactions formed by hydrophobic residues from both the α- and β-domain, which could be important for initiating and driving folding. The molten globule’s 3D topology has been probed by measuring long-range distances between MTSL pairs using Double Electron-Electron Resonance (DEER). Broad distance distributions have been identified between elements of secondary structure, indicative of a fluctuating but compact fold. By contrast, a narrower distance distribution has been measured within one of the major helices, indicative of native-like secondary structure. The surface accessibility of all-Ala α-LA and that of two other variants ([28-111] α-LA and 4SS α-LA) has been probed using solvent PREs obtained using TEMPOL, a paramagnetic co-solute. This has revealed differences in the solvent-exposure of hydrophobic residues due to the removal of disulphide bonds. This method has also identified buried hydrophobic residues that contribute to forming the molten globule’s stable, native-like core.
Supervisor: Redfield, Christina Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.639965  DOI: Not available
Keywords: Biochemistry ; NMR spectroscopy ; Protein folding ; Spectroscopy and molecular structure ; molten globule ; human alpha-lactalbumin ; nuclear magnetic resonance spectroscopy ; electron paramagnetic resonance spectroscopy ; paramagnetic relaxation enhancement ; double electron-electron resonance
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