Site-directed Mutagenesis of Arabidopsis Calmodulin Isoform 2 and Its Application in Detecting Calcium-independent Calmodulin-binding Proteins

Not only calmodulin (CaM) with Ca(2+) regulates the activity of many enzymes and proteins, but also free-CaM (no Ca(2+) bound) and Ca(2+)-independent CaM-binding proteins play roles in plant and animal cells. There is no in vivo method to identify the interaction between free-CaM and Ca(2+)-independent CaM-binding protein (CaMBP). Using site-directed mutagenesis by polymerase chain reaction (PCR), 5 mutant Arabidopsis calmodulin isoform 2 (AtCaM2) genes, mCaM2(1), mCaM2(12), mCaM2(123), mCaM2(124) and mCaM2(1234) were obtained. The mutant mCaM2 encoded glutamine in place of glutamate (E32Q; E68Q; E105Q; E141Q) in one or more EF-hand Ca(2+)-binding motifs of AtCaM2. The recombinant mCaM2 proteins were produced in Escherichia coli, and subsequently separated on SDS-PAGE in the presence of Ca(2+) or EGTA, their electrophoresis mobilities were related with that of mutant EF-hand motifs. (45)Ca(2+) overlay analysis indicated that the more glutamate replaced by glutamine, the lower affinity with Ca(2+) in the mCaM2 proteins. The mCaM2(1234) mutant protein (E32Q; E68Q; E105Q; E141Q) was unable to bind Ca(2+). Using yeast two-hybrid technique with mCaM2(1234) as bait, it was possible to see interaction in Arabidopsis of AtCaM2 with IQD26, a calcium-independent CaM-binding protein. Site-directed mutation of AtCaM2 will aid the research of Ca(2+), CaM and Ca(2+)-independent CaMBPs in plant biological processes.