Calmodulin (CaM) is a ubiquitous calcium sensor protein which binds and activates a variety of enzymes involved in cell signaling pathways. In its calcium loaded state, CaM is extremely resistant to heat denaturation, with a melting temperature (Tm) of around 115°C. In this study, Xenopus laevis CaM was prepared such that the eight phenylalanine residues were substituted with 3-fluorophenylalanine. 19F NMR studies then focused on properties of the hydrophobic core associated with the folding process at temperatures near the regime where the protein is completely folded. Near 70°C, near-UV circular dichroism and 1H NMR-based measurements of protein diffusion rates reveal the onset of a stable, expanded near-native folding intermediate. 19F NMR solvent isotope shifts reveal a gradual loss of water from the hydrophobic core with increasing temperature, until the point at which the near-native intermediate state is attained. At this point, water is observed to enter the hydrophobic core and destabilize the protein. Paramagnetic shifts from dissolved oxygen reveal an increase in oxygen accessibility with temperature until the near-native intermediate is reached, whereupon oxygen solubility decreases. Taken together, we conclude that hydrophobicity of the protein interior increases with temperature, until a dry near-native state is established, whereupon water cooperatively enters and destabilizes the hydrophobic core. 19F CPMG experiments provide a measure of the interconversion between the folded state and the dry near-native intermediate; at higher temperatures, folding rates are on the order of 10,000 Hz. Moreover, as temperature is lowered, folding rates increase, presumably because the effect of off-pathway misfolding events on the exchange process is diminished.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/33562 |
| Date | 27 November 2012 |
| Creators | Thach, William |
| Contributors | Prosser, Robert Scott |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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