X-ray diffraction studies of red tomato nuclease TBN1
T. Kovaľ1,2, J. Dohnálek1,3, P.
Lipovová4, T. Podzimek4, J. Matoušek5, J.
Dušková3, T. Skálová3, A. Štěpánková3, P.
Kolenko3 and J. Hašek3
1Institute of Physics, Academy of Sciences of the Czech Republic, Cukrovarnická 10, Praha 6, Czech Republic,
2Fac. Of Mathematics and Physics, Charles University in Prague, Ke Karlovu 3, Praha 2, Czech Republic,
3Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovského nám. 2, Praha6, Czech Republic
4Institute of Chemical Technology, Technická 5, Praha 6, Czech Republic
5Institute of Plant Molecular Biology, Biology Centre, Academy of
Sciences of the Czech Republic, Branišovská 31, České Budějovice, Czech
Republic
koval@fzu.cz
Bifunctional nuclease TBN1 (accession no. AM23701) from Solanum lycopersicum - red tomato is a Zn2+- dependent plant glycoprotein composed of 277 amino acids with a molecular mass of 31.6 kDa (about 37 kDa when glycosylated). TBN1 belongs to plant nuclease I group and plays a considerable role in specific apoptotic functions and in plant tissue differentiation, vascular system development and stress response [1]. Therefore detailed structural study of this enzyme can provide knowledge applicable in research leading to new ways of cancer, bacterial and viral disease treatment. Nuclease P1 from Penicillium citrinum with 24% sequence identity is probably the closest structural homologue of TBN1, the structure of which is known (PDB ID 1ak0) [2].
Heterologous expression in tobacco leaves yields amounts and quality of the enzyme suitable for structural studies. Crystallization leads to crystals with sufficient quality for X-ray diffraction analysis. First diffraction experiments were performed using in house Gemini Enhanced Ultra diffractometer with the Atlas CCD detector and two different crystal morphologies were identified (orthorhombic and rhombohedral). Datasets for structural analysis were collected at the synchrotron source BESSY, beam line BL14.1, detector MARmosaic CCD 225 and Rigaku rotating anode FR-E+ SuperBright with dual wavelength anode at copper wavelength, CCD detector Saturn A200. Presence of zinc in the protein was confirmed by an absorption edge scan at the synchrotron. A partial MAD dataset was collected at BESSY (Helmholtz-Zentrum Berlin) and attempts to solve the phase problem were performed using the SHELXC, D and E program suite [3].
References
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Acknowledgements.
The work on this project was supported by the Czech Science Foundation, projects no. 310/09/1407, 202/06/0757 and.521/09/1214 and by the European Commission under ELISA grant agreement number 226716 (synchrotron access funding, projects 09.1.81077 and 09.2.90262). We also gratefully acknowledge support from Praemium Academiae of AS CR and Institution research plan AVOZ10100521 of the Institute of Physics AS CR. The authors wish to thank Dr. Uwe Müller of the Helmholtz-Zentrum Berlin, Albert-Einstein-Str. 15 for support at the beam line BL14.1 of Bessy II.