Protein folding pathways can be extraordinarily complex. In this study, circular dichroism (CD) and 19F NMR are used to investigate the folding network of calmodulin, a calcium-binding protein, which is biosynthetically enriched with 3-fluorophenylalanine. In calmodulin’s calcium-loaded state, CD experiments identify the existence of a folding intermediate along a heat-denaturation pathway. In comparison to the native state, 19F NMR solvent isotope shifts reveal decreased accessibility of water to hydrophobic core, whereas O2 paramagnetic shifts show increased hydrophobicity of this folding intermediate. 15N-1H and methyl 13C-1H HSQC NMR spectra demonstrate that this folding intermediate retains a near-native tertiary structure, whose hydrophobic interior is highly dynamic. 19F NMR CPMG relaxation dispersion measurements suggest that this near-native intermediate state is transiently adopted below the temperature associated with its onset. The folding network also involves an unproductive off-pathway intermediate. In contrast, calmodulin’s calcium-free state exhibits a simpler folding process which lacks discernible intermediates.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OTU.1807/42862 |
| Date | 26 November 2013 |
| Creators | Hoang, Joshua Nam |
| Contributors | Prosser, Robert Scott |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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