A site-directed spin labelling study of the human alpha-lactalbumin molten globule
<p>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...
| Main Authors: | , |
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| Format: | Thesis |
| Language: | English |
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2013
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| _version_ | 1826297291988795392 |
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| author | Young, M Matthew Young |
| author2 | Redfield, C |
| author_facet | Redfield, C Young, M Matthew Young |
| author_sort | Young, M |
| collection | OXFORD |
| description | <p>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.</p> |
| first_indexed | 2024-03-07T04:29:21Z |
| format | Thesis |
| id | oxford-uuid:cdc9904d-77cc-4d20-b01d-096bc2b16d72 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T04:29:21Z |
| publishDate | 2013 |
| record_format | dspace |
| spelling | oxford-uuid:cdc9904d-77cc-4d20-b01d-096bc2b16d722022-03-27T07:31:06ZA site-directed spin labelling study of the human alpha-lactalbumin molten globuleThesishttp://purl.org/coar/resource_type/c_db06uuid:cdc9904d-77cc-4d20-b01d-096bc2b16d72Protein foldingNMR spectroscopySpectroscopy and molecular structureBiochemistryEnglishOxford University Research Archive - Valet2013Young, MMatthew YoungRedfield, C<p>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.</p> |
| spellingShingle | Protein folding NMR spectroscopy Spectroscopy and molecular structure Biochemistry Young, M Matthew Young A site-directed spin labelling study of the human alpha-lactalbumin molten globule |
| title | A site-directed spin labelling study of the human alpha-lactalbumin molten globule |
| title_full | A site-directed spin labelling study of the human alpha-lactalbumin molten globule |
| title_fullStr | A site-directed spin labelling study of the human alpha-lactalbumin molten globule |
| title_full_unstemmed | A site-directed spin labelling study of the human alpha-lactalbumin molten globule |
| title_short | A site-directed spin labelling study of the human alpha-lactalbumin molten globule |
| title_sort | site directed spin labelling study of the human alpha lactalbumin molten globule |
| topic | Protein folding NMR spectroscopy Spectroscopy and molecular structure Biochemistry |
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