Structure determination of membrane proteins by solution nuclear magnetic resonance spectroscopy requires that they can be incorporated into detergent micelles in order to ensure that they are maintained in a folded, water-soluble state. However, some detergents appear to disrupt structurally important interactions between transmembrane (TM) helices, while others produce micelle-protein complexes that may be too large for solution NMR. As a result there are a limited number of detergents suitable for solution NMR studies, making the search for better solution conditions one of the remaining challenges for structure elucidation of membrane proteins by this technique. This thesis presents the development and application of methods to address this problem by characterizing detergents for suitability for membrane protein solution NMR. Since the overall size of the membrane protein-detergent complex is one of the major factors that affects the quality of NMR spectra, modified versions of pulsed field gradient (PFG) NMR pulse programs for translational diffusion measurement were set up to estimate the size of these complexes. These methods were applied to a model membrane protein-detergent system allowing an approximate complex size and composition to be determined. 15N relaxation parameters were also measured for this complex and provided evidence that extensive motion of the protein within the micelle can occur to improve the quality of the resulting NMR spectrum. In addition to these experiments that probe complex molecular weight, a model system was also established to help identify detergent characteristics that can promote helix-helix interactions. This system is based on the properties of the single transmembrane segment from the M13 bacteriophage major coat protein (MCPTM) which forms homodimers in both lipid membranes and detergent micelles. Using solution NMR in combination with paramagnetic probes, oligomerization of this peptide under a range of detergent conditions was investigated. The results show that both denaturing and non-denaturing detergents are capable of preserving the TM-helix homodimer interaction. In accord with previous studies on this dimer, results from this study indicate that this peptide exists as a parallel dimeric structure at low concentrations of detergents. Together the methods described in this thesis have the potential to be used in the characterization of a wider range of detergent-protein complexes to assist in the development of new solvents that can ultimately advance the utility of solution NMR of membrane proteins.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/27307 |
| Date | January 2006 |
| Creators | Wu, Yanqiu |
| Publisher | University of Ottawa (Canada) |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 120 p. |
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