Calcium binding properties of recombinant calcium binding protein 40, a major calcium binding protein of lower eukaryote Physarum polycephalum

Calcium binding protein 40 (CBP40) is a Ca2+-binding protein abundant in th e plasmodia of Physarum polycephalum. CBP40 consists four EF-hand domains i n the COOH-terminal half and a putative alpha-helix domain in the NH2-termi nal half. We expressed recombinant proteins of CBP40 in Escherichia coli to investigate its Ca2+-binding properties. Recombinant proteins of CBP40 bou nd 4 mol of Ca2+ with much higher affinity (pCa(1/2) = 6.5) than that of ca lmodulin. When residues 1-196 of the alpha-helix domain were deleted, the a ffinity for Ca2+ decreased to pCa(1/2) = 4.6. A chimeric calmodulin was gen erated by conjugating the alpha-helix domain of CBP40 with calmodulin. The affinity of Ca2+ for the chimeric calmodulin was higher than that for calmo dulin, suggesting that the alpha-helix domain is responsible for the high a ffinity of CBP40 for Ca2+. CBP40 forms large aggregates reversibly in a Ca2 +-dependent manner. A mutant protein with a deletion of NH2-terminal 32 res idues, however, could not aggregate, indicating the importance of these res idues for the aggregation. The aggregation occurs above micromolar levels o f Ca2+ concentration, so it may only occur when CBP40 is secreted out of th e plasmodial cells.