Electrostatic contributions to the binding of Ca2+ in calbindin D9k

Area of Science:

• Biochemistry and Molecular Biology
• Protein-Ligand Interactions
• Calcium-Binding Proteins

Background:

• Calbindin D9k belongs to the calmodulin superfamily of intracellular regulatory proteins.
• Understanding calcium ion (Ca2+) binding is crucial for deciphering cellular signaling pathways.
• Previous studies indicated Ca2+ binding cooperativity in calbindin D9k at low salt concentrations.

Purpose of the Study:

• To investigate the influence of protein surface charges on Ca2+ binding to calbindin D9k.
• To determine the effects of varying electrolyte (KCl) concentrations on Ca2+ binding constants.
• To analyze the impact of specific mutations on Ca2+ binding affinity and cooperativity.

Main Methods:

• Macroscopic and microscopic Ca2+ binding constants were determined using titration experiments.
• Mutations involved replacing surface carboxylates with amides in calbindin D9k.
• 39K and 2D 1H Nuclear Magnetic Resonance (NMR) spectroscopy were employed to assess K+ binding and structural changes.

Main Results:

• Ca2+ binding affinity (K1K2) decreased significantly with certain mutations and increased KCl concentrations, up to a factor of 2.5 x 10^5.
• Positive cooperativity of Ca2+ binding persists at physiological ionic strength (0.15 M KCl), amounting to 5 kJ.mol-1.
• Potassium (K+) binding is weak and does not alter calbindin D9k conformation, indicating unspecific effects of KCl on Ca2+ affinity.

Conclusions:

• Protein surface charges play a significant role in modulating Ca2+ binding affinity.
• Electrolyte concentration influences Ca2+ binding cooperativity and affinity through unspecific electrostatic interactions.
• Mutations affecting surface charges can drastically alter Ca2+ binding properties without significant conformational changes.