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| Hauptseite > Publikationsdatenbank > Analysis of functional domains of Par-4 |
| Hauptseite > Publikationsdatenbank > Analysis of functional domains of Par-4 |
| Dissertation / PhD Thesis/Book | PreJuSER-19996 |
2012
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
Jülich
ISBN: 978-3-89336-766-5
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Abstract: Par-4 is a tumor suppressor protein with pro-apoptotic function. Sequence analysis and secondary structure predictions indicated the presence of coiled coil domain with leucine heptad repeat and a SAC domain with a NLS which is of pharmaceutical interest because it induces apoptosis selectively in cancer cells but not in normal cells. Previously it was shown to be an intrinsically disordered protein. Apparently the full length Par-4 does not fold properly and is subsequently prone to aggregation. Therefore the protein was divided into two functional domains which were investigated separately. The CC domain was purified and crystallized to obtain diffracting crystals. For the determination of experimental phases SeMet labeled CC domain was crystallized. MAD data sets were useful to 2.9 Å. The space group determination was complicated by the presence of merohedral twinning. Also non-merohedral twinning of isomorphous subgroups was investigated. Consequently models were built in three space groups and refined. From the results of the refinements it was concluded based on the free R-value of 34 % that the proper space group is P4$_{3}$. All models showed the presence of parallel running dimeric canonical coiled coil domains with a leucine zipper sub-domain. SAC domain purification was cloned and purified. CD experiments showed the presence of residual structure with predominant random coil profile. Among the prepared phosphorylation mimicking mutants, SAC-T155E was found to have different properties than the native protein. It was shown to be shifted towards the pre-molten globular state by CD spectroscopy. 2D $^{1}$H-$^{15}$N HSQC NMR experiments also showed shifts for some peaks which reflects the change in the surrounding environment of the amino acid residues, proving a weak but consistent change. First crystallization experiments of SAC domain with a Bcl-2 DNA fragment were performed. The observed dimerization behavior of the CC domain and the SAC domain can explain apparently contradicting results reported from in vivo experiments: in the light of these experiments the functional apoptosis inducing unit appears to be the SAC domain dimer which requires the CC domain for dimerization.
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