Calcium binding to complexes of calmodulin and calmodulin binding proteins.
The free energy of coupling for binding of Ca2+ and the calmodulin-sensitive phosphodiesterase to calmodulin was determined and compared to coupling energies for two other calmodulin binding proteins, troponin I and myosin light chain kinase. Free energies of coupling were determined by quantitating binding of Ca2+ to calmodulin complexed to calmodulin binding proteins with Quin 2 to monitor free Ca2+ concentrations. The geometric means of the dissociation constants (-Kd) for Ca2+ binding to calmodulin in the presence of equimolar rabbit skeletal muscle troponin I, rabbit skeletal muscle myosin light chain kinase, and bovine heart calmodulin sensitive phosphodiesterase were 2.1, 1.1, and 0.55 microM. The free-energy couplings for the binding of four Ca2+ and these proteins to calmodulin were -4.48, -6.00, and -7.64 kcal, respectively. The Ca2+-independent Kd for binding of the phosphodiesterase to calmodulin was estimated at 80 mM, indicating that complexes between calmodulin and this enzyme would not exist within the cell under low Ca2+ conditions. The large free-energy coupling values reflect the increase in Ca2+ affinity of calmodulin when it is complexed to calmodulin binding proteins and define the apparent positive cooperativity for Ca2+ binding expected for each system. These data suggest that in vitro differences in free-energy coupling for various calmodulin-regulated enzymes may lead to differing Ca2+ sensitivities of the enzymes.
D000634:Aminoquinolines; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002417:Cattle; D005456:Fluorescent Dyes; D007700:Kinetics; D009206:Myocardium / Q000201:enzymology; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D013050:Spectrometry, Fluorescence
D000634:Aminoquinolines; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D005456:Fluorescent Dyes; D010727:Phosphoric Diester Hydrolases; C035739:Quin2; D002118:Calcium