title: Calcium binding properties of calcium dependent protein kinase 1 (CaCDPK1) from Cicer arietinum.
abstract: Calcium plays a crucial role as a secondary messenger in all aspects of plant growth, development and survival. Calcium dependent protein kinases (CDPKs) are the major calcium decoders, which couple the changes in calcium level to an appropriate physiological response. The mechanism by which calcium regulates CDPK protein is not well understood. In this study, we investigated the interactions of Ca(2+) ions with the CDPK1 isoform of Cicer arietinum (CaCDPK1) using a combination of biophysical tools. CaCDPK1 has four different EF hands as predicted by protein sequence analysis. The fluorescence emission spectrum of CaCDPK1 showed quenching with a 5 nm red shift upon addition of calcium, indicating conformational changes in the tertiary structure. The plot of changes in intensity against calcium concentrations showed a biphasic curve with binding constants of 1.29 μM and 120 μM indicating two kinds of binding sites. Isothermal calorimetric (ITC) titration with CaCl2 also showed a biphasic curve with two binding constants of 0.027 μM and 1.7 μM. Circular dichroism (CD) spectra showed two prominent peaks at 208 and 222 nm indicating that CaCDPK1 is a α-helical rich protein. Calcium binding further increased the α-helical content of CaCDPK1 from 75 to 81%. Addition of calcium to CaCDPK1 also increased fluorescence of 8-anilinonaphthalene-1-sulfonic acid (ANS) indicating exposure of hydrophobic surfaces. Thus, on the whole this study provides evidence for calcium induced conformational changes, exposure of hydrophobic surfaces and heterogeneity of EF hands in CaCDPK1.
authors: Department of Biochemistry, Indian Institute of Science, Bangalore 560012, Karnataka 560012, India. Electronic address: ajaydixit304@gmail.com.
authors: Ajay Kumar
authors: AK
authors: Dixit
authors: Department of Biochemistry, Indian Institute of Science, Bangalore 560012, Karnataka 560012, India.
authors: Chelliah
authors: C
authors: Jayabaskaran
mesh_terms: D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism*; D002151:Calorimetry; D029863:Cicer / Q000201:enzymology*; D002942:Circular Dichroism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D017871:Calcium-Calmodulin-Dependent Protein Kinases
chemical_list: D002118:Calcium; D010940:Plant Proteins; D011494:Protein Kinases; D011994:Recombinant Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases
keywords: Abiotic stress; Calcium dependent protein kinases; Conformational change; Fluorescence; Isothermal titration calorimetry