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-te...

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Bibliographic Details
Published in:The Journal of biological chemistry 1991-05, Vol.266 (13), p.8050-8054
Main Authors: LINSE, S, HELMERSSON, A, FORSEN, S
Format: Article
Language:English
Subjects:
Analytical, structural and metabolic biochemistry
Animals
Binding and carrier proteins
Binding, Competitive
Biological and medical sciences
calcium
Calcium - metabolism
calmodulin
Calmodulin - metabolism
Calmodulin - ultrastructure
Cattle
Chelating Agents - metabolism
Egtazic Acid - metabolism
Fundamental and applied biological sciences. Psychology
Hydrogen-Ion Concentration
Magnetic Resonance Spectroscopy
Peptide Fragments - metabolism
Potassium - metabolism
Potassium Chloride
Protein Conformation
Proteins
Thermodynamics
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Summary: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.
ISSN:0021-9258
1083-351X
DOI:10.1016/s0021-9258(18)92938-8