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Complementary biochemical approaches applied to the identification of plastidial calmodulin-binding proteins.
- Source :
-
Molecular bioSystems [Mol Biosyst] 2013 Jun; Vol. 9 (6), pp. 1234-48. Date of Electronic Publication: 2013 Apr 03. - Publication Year :
- 2013
-
Abstract
- Ca(2+)/Calmodulin (CaM)-dependent signaling pathways play a major role in the modulation of cell responses in eukaryotes. In the chloroplast, few proteins such as the NAD(+) kinase 2 have been previously shown to interact with CaM, but a general picture of the role of Ca(2+)/CaM signaling in this organelle is still lacking. Using CaM-affinity chromatography and mass spectrometry, we identified 210 candidate CaM-binding proteins from different Arabidopsis and spinach chloroplast sub-fractions. A subset of these proteins was validated by an optimized in vitro CaM-binding assay. In addition, we designed two fluorescence anisotropy assays to quantitatively characterize the binding parameters and applied those assays to NAD(+) kinase 2 and selected candidate proteins. On the basis of our results, there might be many more plastidial CaM-binding proteins than previously estimated. In addition, we showed that an array of complementary biochemical techniques is necessary in order to characterize the mode of interaction of candidate proteins with CaM.
- Subjects :
- Arabidopsis Proteins metabolism
Calcium chemistry
Calcium metabolism
Calmodulin chemistry
Calmodulin metabolism
Calmodulin-Binding Proteins chemistry
Calmodulin-Binding Proteins genetics
Gene Expression Profiling
Phosphotransferases (Alcohol Group Acceptor) analysis
Phosphotransferases (Alcohol Group Acceptor) chemistry
Plant Leaves
Plant Proteins metabolism
Protein Binding
Signal Transduction
Arabidopsis metabolism
Calmodulin-Binding Proteins metabolism
Chloroplasts metabolism
Proteome analysis
Spinacia oleracea metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1742-2051
- Volume :
- 9
- Issue :
- 6
- Database :
- MEDLINE
- Journal :
- Molecular bioSystems
- Publication Type :
- Academic Journal
- Accession number :
- 23549413
- Full Text :
- https://doi.org/10.1039/c3mb00004d