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| Hauptseite > Publikationsdatenbank > Kristallisation und NMR-spektroskopische Untersuchungen an dem Twisted Dwarf Protein aus Arabidopsis thaliana |
| Hauptseite > Publikationsdatenbank > Kristallisation und NMR-spektroskopische Untersuchungen an dem Twisted Dwarf Protein aus Arabidopsis thaliana |
| Dissertation / PhD Thesis/Book | PreJuSER-5949 |
2003
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
Jülich
Please use a persistent id in citations: http://hdl.handle.net/2128/86
Report No.: Juel-4023
Abstract: The Twisted-Dwarf (TWD)-Protein from Arabidopsis thaliana belongs to the class of FK506 binding proteins (FKBP). Proteins of that class are offen involved in formation of multi-protein complexes. TWD-Plants exhibit a pleiotropic phenotype, that shows both strong effects an morphology and developmental cycle of the plant. In combination with interaction studies of cellular partners, the threedimensional structure ofthe TWD-Protein may yield information an processes, that are important for the architecture of the plants. In the present work, protocols for heterologous expression of the TWD-Protein in E.coli and purification of the recombinant protein were developed. By using standard biomolecular techniques, an affinity tag was fused to the C-terminus ofthe TWD-Protein. Sequential IMAC, gelfiltration, and ion exchange chromatography was applied for purification. Although both purity and amount of recombinant protein would have been sufficient for crystallization, it was not possible to concentrate the protein to more than 2 mg/ml, which is sufficiently lower than the typical threshold of 5 mg/ml. For that reason, all subsequent crystallization trials were conducted with two shortened constructs of the TWD-Protein: TWD without membrane anchor (TWD-MA) and the N-terminal FKBP-Domain (TWD12D), kindly provided by T.Kamphausen (Max Planck Reserach Unit for Enzymology of Protein Folding, Halle/Saale). For succesfull crystallization ofTWD the presence ofthe precipitation agent sodiumformate was crucial. In contrast, TWD12D did crystallize in the presence of either ammoniumsulfate or ammoniumphosphate. In all cases the crystallization time was ranging from about 3 month to one year. Native and heavy atom X-ray synchrotron datasets (ES-F, Grenoble, France) were collected for TWD-MA. First X-ray-diffraction patterns of the TWD12D-Crystals Show a good diffraction quality. Resolution of about 2$\mathring{A}$ can be expected for these crystals with X-ray synchrotron radiation. Future goals are the calculation of the high resolution TWD-MA structure based an the available data, and recording of X-ray diffraction data an a synchrotron source for TWD12D followed by structure calculation. Additional crystallization attempts will be directed at shortening the crystallization time. In addition to the X-ray crystallographic analysis NMR-based 3D-Structure determination of TVVD12D was initiated. TWD12D was labeled with NMR-active isotopes. A complete set of standard threedimensional high resolution NMR-experiments for resonance assignment was recorded and the assigment process was started. Initial results from both techniques indicate, that the 40 N-terminal residues are not structured. Therefore it is proposed to perform a complete structure analysis an a new construct, which is lacking these 40 residues. Using such a shortened construct might aid both the crystallization process and the NMR-Data analysis.
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