Calcium binding to calmodulin and its globular domains.
The macroscopic Ca(2+)-binding constants of bovine calmodulin have been determined from titrations with Ca2+ in the presence of the chromophoric chelator 5,5'-Br2BAPTA in 0, 10, 25, 50, 100, and 150 mM KCl. Identical experiments have also been performed for tryptic fragments comprising the N-terminal and C-terminal domains of calmodulin. These measurements indicate that the separated globular domains retain the Ca2+ binding properties that they have in the intact molecule. The Ca2+ affinity is 6-fold higher for the C-terminal domain than for the N-terminal domain. The salt effect on the free energy of binding two Ca2+ ions is 20 and 21 kJ. mol-1 for the N- and C-terminal domain, respectively, comparing 0 and 150 mM KCl. Positive cooperativity of Ca2+ binding is observed within each globular domain at all ionic strengths. No interaction is observed between the globular domains. In the N-terminal domain, the cooperativity amounts to 3 kJ.mol-1 at low ionic strength and greater than or equal to 10 kJ.mol-1 at 0.15 M KCl. For the C-terminal domain, the corresponding figures are 9 +/- 2 kJ.mol-1 and greater than or equal to 10 kJ.mol-1. Two-dimensional 1H NMR studies of the fragments show that potassium binding does not alter the protein conformation.
Linse
S
S
Physical Chemistry 2, Lund University, Sweden.
Helmersson
A
A
Forsén
S
S
D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism* / Q000648:ultrastructure; D002417:Cattle; D002614:Chelating Agents / Q000378:metabolism; D004533:Egtazic Acid / Q000378:metabolism / Q000031:analogs & derivatives; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D010446:Peptide Fragments / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D011189:Potassium Chloride; D011487:Protein Conformation; D013816:Thermodynamics
D002147:Calmodulin; D002614:Chelating Agents; D010446:Peptide Fragments; C053459:5,5'-dibromo-1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D004533:Egtazic Acid; D011189:Potassium Chloride; D011188:Potassium; D002118:Calcium