FIGURE 5.

Formation of 1:2 CaM·Orai1-CMBD complex revealed by size exclusion chromatography. A, chromatograms of wild type CaM/Orai1-CMBD mixtures. B, chromatograms of CaM mutant/Orai1-CMBD mixtures. The two calcium binding sites of CaM-N were abolished in the CaM mutant so that only its C-lobe can bind Orai1-CMBD. The Orai1-CMBD-Trx fusion protein, CaM (or CaM mutant), and Orai1-CMBD-Trx/CaM mixture at 1:1, 2:1, or 4:1 molar ratios were individually subjected to a Superdex 200 column. The molecular weights of proteins in each elution peak were calculated from the elution volumes as described under “Experimental Procedures.” The results show that two Orai1-CMBDs bind to one CaM.