Characterisation of the effects of cosolutes on the
stability of H-bonds in proteins by NMR spectroscopy.
Doctoral thesis, UCL (University College London).
The strength of the hydrogen bond (H-bond) in biomacromolecules is significantly weaker compared to covalent bonds. Consequently, H-bonds readily form and break under physiological conditions. This property explains the key role H-bonds play in the stabilisation of biological macromolecules and as participants in many enzymatic reactions. Ubiquitin is often used in NMR experiments as a model protein due to its high solubility and stability. In addition, the folding of ubiquitin has been shown to follow a simple two-state process. As such, ubiquitin is an ideal protein to examine the subtle effects of different solvent conditions on secondary structure. In this research project a variety of NMR parameters are used to characterise the changes in the character (i.e. geometry/strength) of the H-bonds in ubiquitin under different physicochemical conditions. In particular, the research project focuses on measuring H-bond scalar couplings (HBCs), and correlating the HBCs to other NMR parameters such as the amide 1H isotropic chemical shift and 15N relaxation data. The NMR data is complimented by circular dichroic data that provides a “global” picture of the protein structural state under the various co-solute conditions examined. The H-bonds properties of ubiquitin where observed over a range of four temperatures 15 to 60 ºC in the presence and absence of 1.5 M trimethylamine-N-oxide (TMAO). TMAO is an organic osmolyte produced by certain species of fish to counteract high concentrations of urea found in their cells. In the absence of TMAO a global decrease in h3JNC’ couplings were observed with increasing temperatures. This observation indicates a thermal expansion of the protein as the temperature increased. The weakening of the HBCs correlated with an upfield shift of the amide 1HN chemical shift and a downfield shift of the donor 15N chemical shift. The NMR results were supported by CD data in which a global decrease in ellipticity values was observed as the temperature was increased. In the presence of 1.5 M TMAO the average decrease in h3JNC’ couplings between 15 and 60 ºC were smaller (0.075 ± 0.001 Hz) compared to value 0.1 ± 0.001 Hz recorded in the absence of TMAO. Using these HBC values, the thermal expansion coefficient in the absence of TMAO was 3.3 (±0.2) 10-4 K–1, whereas in the presence of TMAO a value of 2.2 (±0.2) 10-4 K–1was calculated. The slower rate of thermal expansion of H-bonds in ubiquitin in the presence of TMAO indicates that this co-solute slows the thermal denaturation of ubiquitin and shifts the melting point of ubiquitin to a higher value. In the presence of TMAO, the correlation between the HBCs and the 1H and 15N chemical shifts is significantly weaker compared to data recorded in the absence of TMAO. Presumably, the presence of TMAO not only indirectly influences the H-bond character, but also the chemical environment of the donor amide group. This is not unexpected since the HBCs are solely influenced by the H-bond geometry, whereas the chemical environment surrounding the donor group nuclei influences the 1H and 15N chemical shifts. Urea and Guanidinium chloride (Gdn.HCl) are denaturants that have been used extensively in protein folding and stability studies. The H-bonds in ubiquitin where observed over different Gdn.HCl concentrations from 0 to 3.0 M. An increase in some of the h3JNC’ couplings were observed at Gdn.HCl concentrations below 1.5 M. This observation follows previous research that has shown that the salt stabilising characteristics of Gdn.HCl prevail at low concentrations. At 3 M Gdn.HCl a global decrease in HBCs was observed. Due to the two-state folding process of ubiquitin, the acquisition of NMR data beyond a Gdn.HCl concentration of < 3.0 M gave rise to second sets of peaks: one set of peaks represented the native state of ubiquitin, while the second set of peaks represents ubiquitin in the unfolded state. As such, the recording of data beyond this concentration would not provide any further insight into the denaturation of ubiquitin. At the concentrations examined (i.e. < 3.0 M), this study provided insights into the influence of Gdn.HCl on the stability and character of Hbonds in ubiquitin. In a similar series of NMR experiments, the effect of urea on the structure of ubiquitin was examined at concentrations ranging between 0 and 3 M.
|Title:||Characterisation of the effects of cosolutes on the stability of H-bonds in proteins by NMR spectroscopy|
|Open access status:||An open access version is available from UCL Discovery|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Life Sciences > Biosciences (Division of) > Structural and Molecular Biology|
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