Positive cooperative binding of calcium to bovine brain calmodulin.
Equilibrium dialysis measurements of the binding of Ca2+ to calmodulin have confirmed the existence of four high affinity Ca2+-binding sites (Kd between 3 X 10(-6) and 2 X 10(-5) M). In the presence of 3 mM Mg2+, the dissociation constants for Ca2+ are increased two- to fourfold (Kd between 5 X 10(-6) and 4 X 10(-5) M). Positive cooperativity of Ca2+ binding was observed at low Ca2+ concentrations with Hill coefficients of 1.33 and 1.22 in the absence and presence of 3 mM Mg2+, respectively. The positive cooperativity is compatible with the steepness of the Ca2+ dependence of the conformational transition associated with the binding of 2 mol of Ca2+/mol of calmodulin. This conformational change, which affects the environment of the aromatic residues of calmodulin as measured by UV absorption and near-UV circular dichroism spectroscopy, is not the result of a monomer-dimer equilibrium mediated by Ca2+. Binding of Ca2+ to calmodulin is believed to occur by a sequential mechanism generating at least four different conformers of the protein and its free and liganded states. Even though the major conformational change is almost complete upon binding of 2 mol of Ca2+/mol of calmodulin, the activation of cyclic nucleotide phosphodiesterase measured in the presence of limiting concentrations of calmodulin suggests that a calmodulin Ca3-42+ complex is required for interaction of calmodulin with the enzyme. As expected, on the basis of the strong affinity of the enzyme for the calmodulin x Ca2+ complex (Kd = 1-3 X 10(-9) M), the Ca2+ dependence of phosphodiesterase activation is highly cooperative and leads to a sharp threshold of Ca2+ concentration for control of enzyme activity.
D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D002942:Circular Dichroism; D008433:Mathematics; D011485:Protein Binding; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D002118:Calcium