Expression and characterization of Calmodulin and Calmodulin-like proteins from rice Oryza sativa L.

Abstract

Calmodulin (CaM) is a small calcium receptor protein that contains Ca2+-binding EF hand motifs. When CaMs bind Ca2+ ions, they undergo conformational changes revealing their hydrophobic surfaces, which bind to and alter the activities of target proteins that affect physiological responses to the vast array of specific stimuli received by plants. Previously, a large family of Cam and calmodulin-like (CML) genes has been identified in Oryza sativa L. Here, we examined properties of the purified recombinant proteins encoded by 11 OsCam and OsCML genes. Three OsCML genes, OsCML9, OsCML11 and OsCML13 were successfully cloned. The recombinant plasmids of these genes and eight other previously cloned genes of OsCam and OsCML were constructed in pET-21a (+) vector and introduced into E. coli BL21 (DE3). After protein production was induced by IPTG, it was shown that all OsCaM and OsCML proteins except OsCML9 could be purified by Ca2+-dependent Phenyl-Sepharose hydrophobic chromatography. By electrophoretic mobility shift, most of them exhibited these characteristic when incubated with Ca2+ indicating that they are functional Ca2+-binding proteins. To examine possible differential target binding, their ability to bind the peptide derived from CaM kinase II was assessed. All OsCaM proteins (OsCaM1, OsCaM2, and OsCaM3) displayed a peptide-protein complex but all OsCML proteins did not form complexes with CaMKII peptides. Changes in their secondary structure upon Ca2+-binding through measurements by Circular dichroism (CD) were determined. All OsCaM and some OsCML proteins displayed a large increase in the molar elipticity when compared with the value in the absence of Ca2+, which indicates that the helical content was highly increased in these proteins upon Ca2+ binding. The ANS fluorescence spectroscopy measurement showed that all proteins examined exhibited a change in the ANS spectrum as the λmax for emission spectrum decreased and the fluorescence intensity increased in the presence of Ca2+. These changes indicate that all proteins bind Ca2+ and expose hydrophobic patches on their surfaces. These results suggest that OsCMLs represent novel sensor proteins whose modes of action are probably different from or overlapping with those of OsCaMs and possibly serve distinct roles and will help us further investigate the molecular functions and physiological significance of these proteins.