Binding of Ca2+ to calbindin D9k: structural stability and function at high salt concentration

Area of Science:

• Biochemistry
• Structural Biology
• Computational Biology

Background:

• Calbindin D9k is a calcium-binding protein crucial for cellular calcium homeostasis.
• Understanding the electrostatic contributions to calcium binding is vital for protein function analysis.

Purpose of the Study:

• To quantify the effect of electrostatic interactions on calcium binding affinity and cooperativity in calbindin D9k.
• To evaluate the validity of dielectric continuum models for protein electrostatics.

Main Methods:

• Site-directed mutagenesis to neutralize negative charges near calcium binding sites.
• Determination of calcium binding constants at varying KCl concentrations (2 mM to 1 M).
• Two-dimensional 1H NMR spectroscopy to assess structural integrity.
• Monte Carlo simulations and Kirkwood-Tanford formula for theoretical analysis.

Main Results:

• High salt concentrations significantly reduced cooperativity of calcium binding to calbindin D9k, indicating strong dependence on electrostatic interactions.
• No significant structural changes in calbindin D9k were observed at high salt concentrations, confirmed by NMR data.
• Excellent agreement was found between experimental data and Monte Carlo simulations for binding constants across a wide range.

Conclusions:

• Electrostatic interactions play a critical role in the cooperativity of calcium binding to calbindin D9k.
• Dielectric continuum models accurately predict electrostatic effects in protein-ligand interactions.
• Salt concentration serves as a controllable method to modulate electrostatic interactions without altering protein structure.