Charakterisierung eines zyklisch Nukleotid-gesteuerten Ionenkanals aus Mesorhizobium loti

Novak, Kerstin

Dissertation / PhD Thesis/Book

Charakterisierung eines zyklisch Nukleotid-gesteuerten Ionenkanals aus Mesorhizobium loti

4224

Novak, K. (Corresponding author)FZJ*

2006
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag Jülich

Jülich : Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag, Berichte des Forschungszentrum Jülich 4224, VII, 117 S. (2006) = Köln, Univ., Diss., 2006

Please use a persistent id in citations: http://hdl.handle.net/2128/2456

Report No.: Juel-4224

Abstract: Ion channels control the flow of ions across cellular membranes. They play a crucial role in many biological processes. Cyclic nucleotide-gated ion channels (CNG- and HCN-channels) belong to the family of ligand-gated ion channels which are regulated by cAMP and cGMP. While the physiological function of these channels is well investigated, only little is known about their structure and molecular mechanism of opening (“gating”). Homologous bacterial ion channels offer a novel basis to investigate the “gating”-mechanism, because they can be expressed in high quantities in bacteria and, therefore, are applicable for structural studies. In this work biochemical and biophysical studies were carried out on the cyclic nucleotidegated ion channel (MlCNG) from the bacterium Mesorhizobium loti. The expression of the MlCNG protein in E. coli was optimized to obtain high yield. The protein was purified via affinity chromatography to enable functional and structural studies. In addition, monoclonal antibodies were raised against the MlCNG protein. The antibodies detect the channel protein in the Western Blot as well as in immunocytochemical stainings. A construct (MlCNBD) was cloned, which only contains the c-terminal cyclic nucleotide-binding domain of the MlcNMP channel. The MlCNBD protein was expressed in bacteria and cAMP-bound and cAMP-free forms of the protein were purified for structural and ligand binding studies. Preliminary NMR measurements to identify the structure of the MlCNBD protein were performed successfully. The binding affinities of MlCNG and MlCNBD for cyclic nucleotides were determined by isothermal titration calorimetry. Chemical cross linking analysis shows that MlCNG as well as MlCNBD form tetrameric complexes.

Note: Record converted from JUWEL: 18.07.2013; Record converted from VDB: 12.11.2012
Note: Köln, Univ., Diss., 2006

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