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Title: Structural studies of Xenopus laevis Quaking protein QUA1 domain
Author: Ali, M.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2010
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A recent report showed that a specific point mutation of glutamate to glycine (E48G) in the QUA1 domain affects both the oligomerisation and the RNA-binding properties of the murine Quaking homologue (QkI). The mutation has also been shown to be embryonic lethal in mice at around day 10 of gestation. The aims of this thesis were to investigate the structural features of QUA1 domain and explain why E48G mutants fail to dimerize. The first objective was pursued by expression and purification of the Xenopus laevis Quaking homologue (pXqua) QUA1 motif, followed by biophysical characterization and structural studies using solution-state nuclear magnetic resonance (NMR) spectroscopy. The second objective was addressed by biophysical characterization of an E48G mutant of the QUA1 domain, prepared using site directed mutagenesis. The QUA1 dimer was shown to comprise two α-helical hairpins assembling via an unusually small and polar protein-protein interface. Based on this structure, we proposed that the properties of the E48G mutant could be explained by disruption of salt-bridge interactions, either between E43 and R46 of the same protomer or R38 of the other protomer. This hypothesis was tested by generating a series of QUA1 mutants by site directed mutagenesis and analyzing them using analytical SEC, both as cleaved proteins and as MBP fusion proteins. Moreover, a separate mutational analysis was performed to compare the pXqua QUA1 domain with the corresponding regions in other STAR/GSG proteins. Sequence alignment of the QUA1 region showed proline residue in the predicted helix α1 of SAM68 and SF1. Corresponding mutations were made in pXqua QUA1 domain and the effect was investigated by studying their oligomerisation.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available