Structural characterisation of the human alpha-lactalbumin molten globule at high temperature.
Molten globules are partially folded forms of proteins thought to be general intermediates in protein folding. The 15N-1H HSQC NMR spectrum of the human alpha-lactalbumin (alpha-LA) molten globule at pH 2 and 20 degrees C is characterised by broad lines which make direct study by NMR methods difficu...
| Príomhchruthaitheoirí: | , |
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| Formáid: | Journal article |
| Teanga: | English |
| Foilsithe / Cruthaithe: |
2003
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| _version_ | 1826296438594732032 |
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| author | Ramboarina, S Redfield, C |
| author_facet | Ramboarina, S Redfield, C |
| author_sort | Ramboarina, S |
| collection | OXFORD |
| description | Molten globules are partially folded forms of proteins thought to be general intermediates in protein folding. The 15N-1H HSQC NMR spectrum of the human alpha-lactalbumin (alpha-LA) molten globule at pH 2 and 20 degrees C is characterised by broad lines which make direct study by NMR methods difficult; this broadening arises from conformational fluctuations throughout the protein on a millisecond to microsecond timescale. Here, we find that an increase in temperature to 50 degrees C leads to a dramatic sharpening of peaks in the 15N-1H HSQC spectrum of human alpha-LA at pH 2. Far-UV CD and ANS fluorescence experiments demonstrate that under these conditions human alpha-LA maintains a high degree of helical secondary structure and the exposed hydrophobic surfaces that are characteristic of a molten globule. Analysis of the H(alpha), H(N) and 15N chemical shifts of the human alpha-LA molten globule at 50 degrees C leads to the identification of regions of native-like helix in the alpha-domain and of non-native helical propensity in the beta-domain. The latter may be responsible for the observed overshoot in ellipticity at 222 nm in kinetic refolding experiments. |
| first_indexed | 2024-03-07T04:16:20Z |
| format | Journal article |
| id | oxford-uuid:c98c2bae-0f18-40a9-83fa-6e4e9090c563 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T04:16:20Z |
| publishDate | 2003 |
| record_format | dspace |
| spelling | oxford-uuid:c98c2bae-0f18-40a9-83fa-6e4e9090c5632022-03-27T06:59:53ZStructural characterisation of the human alpha-lactalbumin molten globule at high temperature.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:c98c2bae-0f18-40a9-83fa-6e4e9090c563EnglishSymplectic Elements at Oxford2003Ramboarina, SRedfield, CMolten globules are partially folded forms of proteins thought to be general intermediates in protein folding. The 15N-1H HSQC NMR spectrum of the human alpha-lactalbumin (alpha-LA) molten globule at pH 2 and 20 degrees C is characterised by broad lines which make direct study by NMR methods difficult; this broadening arises from conformational fluctuations throughout the protein on a millisecond to microsecond timescale. Here, we find that an increase in temperature to 50 degrees C leads to a dramatic sharpening of peaks in the 15N-1H HSQC spectrum of human alpha-LA at pH 2. Far-UV CD and ANS fluorescence experiments demonstrate that under these conditions human alpha-LA maintains a high degree of helical secondary structure and the exposed hydrophobic surfaces that are characteristic of a molten globule. Analysis of the H(alpha), H(N) and 15N chemical shifts of the human alpha-LA molten globule at 50 degrees C leads to the identification of regions of native-like helix in the alpha-domain and of non-native helical propensity in the beta-domain. The latter may be responsible for the observed overshoot in ellipticity at 222 nm in kinetic refolding experiments. |
| spellingShingle | Ramboarina, S Redfield, C Structural characterisation of the human alpha-lactalbumin molten globule at high temperature. |
| title | Structural characterisation of the human alpha-lactalbumin molten globule at high temperature. |
| title_full | Structural characterisation of the human alpha-lactalbumin molten globule at high temperature. |
| title_fullStr | Structural characterisation of the human alpha-lactalbumin molten globule at high temperature. |
| title_full_unstemmed | Structural characterisation of the human alpha-lactalbumin molten globule at high temperature. |
| title_short | Structural characterisation of the human alpha-lactalbumin molten globule at high temperature. |
| title_sort | structural characterisation of the human alpha lactalbumin molten globule at high temperature |
| work_keys_str_mv | AT ramboarinas structuralcharacterisationofthehumanalphalactalbuminmoltenglobuleathightemperature AT redfieldc structuralcharacterisationofthehumanalphalactalbuminmoltenglobuleathightemperature |