Figure 3.

Targeted mutagenesis identifies essential residues required for CaM-binding properties of ceQORH. A, scheme of the residues selected for targeted mutagenesis of the ceQORH sequence. The CAM-binding region in the ceQORH sequence is deduced from successive deletions of ceQORH domains (see Fig. 2). B, SDS-PAGE and CaM overlay. Lanes Ec, purified recombinant QOR from E. coli K12 used as a negative control; lanes M, prestained protein molecular weight markers; lanes 1, purified recombinant WT form of ceQORH from A. thaliana used as a positive control; lanes 10, purified mutagenized form of Arabidopsis ceQORH mutant 1; lanes 11, purified mutagenized form of Arabidopsis ceQORH mutant 2; lanes 12, purified mutagenized form of Arabidopsis ceQORH mutant 3. Note that, in Mut2, mutagenesis of only three residues is sufficient to abolish interaction of ceQORH with CaM.