Calcium binding to calmodulin and its globular domains

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Chemistry

Background:

• Calmodulin (CaM) is a crucial calcium-binding protein involved in numerous cellular processes.
• Understanding CaM's calcium-binding properties is essential for deciphering its regulatory functions.
• Previous studies have investigated CaM's structure-function relationship, but domain-specific binding affinities require further elucidation.

Purpose of the Study:

• To determine the macroscopic Ca(2+)-binding constants of bovine calmodulin and its tryptic fragments.
• To investigate the Ca(2+) binding properties of the N-terminal and C-terminal domains of calmodulin independently.
• To assess the influence of ionic strength on Ca(2+) binding and cooperativity within each domain.

Main Methods:

• Macroscopic Ca(2+)-binding constants were determined using Ca(2+) titrations with the chromophoric chelator 5,5'-Br2BAPTA.
• Experiments were conducted at various KCl concentrations (0-150 mM) to evaluate salt effects.
• Two-dimensional 1H NMR studies were employed to assess the impact of potassium binding on protein conformation.

Main Results:

• Separated N-terminal and C-terminal calmodulin domains retain Ca(2+) binding properties similar to the intact molecule.
• The C-terminal domain exhibits a 6-fold higher Ca(2+) affinity compared to the N-terminal domain.
• Positive cooperativity of Ca(2+) binding is observed within each domain, with varying degrees depending on ionic strength; no inter-domain interaction was detected.

Conclusions:

• The globular domains of calmodulin function independently in Ca(2+) binding.
• Ionic strength significantly influences the salt effect on Ca(2+) binding free energy and cooperativity.
• Potassium binding does not induce conformational changes in the calmodulin fragments, suggesting domain-specific Ca(2+) regulation.