This title appears in the Scientific Report :
2007
Please use the identifier:
http://hdl.handle.net/2128/2458 in citations.
Bindungsstudien an einem bakteriellen zyklisch Nukleotid-gesteuerten Ionenkanal
Bindungsstudien an einem bakteriellen zyklisch Nukleotid-gesteuerten Ionenkanal
Ion channels control the flow of ions across cellular membranes. They play a crucial role in many biological processes. CNG ($\underline{c}$yclic $\underline{n}$ucleotide-$\underline{g}$ated) channels belong to the family of ligand-gated ion channels which are regulated by cAMP and cGMP. While the p...
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Personal Name(s): | Grüter, Bärbel (Corresponding author) |
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Contributing Institute: |
Zelluläre Biophysik; INB-1 |
Imprint: |
Jülich
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
2007
|
Physical Description: |
IX, 123 p. |
Dissertation Note: |
Köln, Univ., Diss., 2006 |
Document Type: |
Book Dissertation / PhD Thesis |
Research Program: |
Funktion und Dysfunktion des Nervensystems |
Series Title: |
Berichte des Forschungszentrums Jülich
4240 |
Subject (ZB): | |
Link: |
OpenAccess |
Publikationsportal JuSER |
Ion channels control the flow of ions across cellular membranes. They play a crucial role in many biological processes. CNG ($\underline{c}$yclic $\underline{n}$ucleotide-$\underline{g}$ated) 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 examined, only little is known about their molecular mechanism of opening (“gating”). Homologous bacterial ion channels offer a novel basis to explore the “gating”-mechanism, because they can be expressed in high quantities in bacteria and, therefore, are applicable for biochemical studies. In this work, biochemical and biophysical studies were performed on the cyclic nucleotide-gated ion channel (MlCNG) from the bacterium Mesorhizobium loti and its isolated cyclic nucleotide-binding domain (CNBD). The CNBD protein was expressed in Escherichia coli (E. coli) and purified in a cAMP-free form. I determined the binding affinities for cAMP, cGMP and some 8-substituted analogs via isothermal titration calorimetry (ITC) and, alternatively with a fluorescence assay. The binding affinities for cAMP and cGMP are in the submicromolar range. cAMP binds to the CNBD with a five fold higher affinity than cGMP. The MlCNG protein was expressed in E. coli as well and purified via affinity chromatography. The oligomeric running behaviour of the MlCNG protein was compared with that of the KcsA channel from Streptomyces lividans. The binding affinity for cAMP was determined via ITC, the affinities for some other cyclic nucleotides were obtained from the fluorescence assay. The K$_{D}$ value for cAMP is about four times higher for the full-length channel than for the isolated binding domain. |