Calbindin D(9k): a protein optimized for calcium binding at neutral pH.

Area of Science:

• Biochemistry
• Structural Biology
• Protein-Ligand Interactions

Background:

• EF-hand proteins are crucial calcium-binding proteins involved in various cellular processes.
• Calcium ion (Ca2+) binding is mediated by electrostatic interactions with negatively charged protein residues.
• Understanding the pH-dependence of Ca2+ binding is vital for elucidating protein function in physiological conditions.

Purpose of the Study:

• To investigate the pH dependence of calcium ion binding by calbindin D(9k).
• To correlate observed binding changes with known pK(a) values of ionizable groups in calbindin D(9k).
• To elucidate the role of electrostatic interactions and protein charge in pH-modulated Ca2+ affinity.

Main Methods:

• Experimental determination of Ca2+ binding affinity across a range of pH values.
• Utilizing calbindin D(9k) with known pK(a) values for precise data interpretation.
• Employing Monte Carlo simulations to model electrostatic interactions and protein charge effects.

Main Results:

• Calcium binding affinity for calbindin D(9k) remains constant between pH 7 and 9, showing maximum affinity.
• A significant decrease in Ca2+ binding affinity occurs below pH 7.
• The pH dependence is characterized by the involvement of two acidic groups with a pK(a) of 6.6, impacting overall protein charge and binding site electrostatics.

Conclusions:

• The pH-dependent reduction in calcium binding is primarily driven by protonation of acidic residues, altering electrostatic interactions.
• Both the immediate binding site and the overall protein charge are affected by pH changes, influencing Ca2+ affinity.
• Calbindin D(9k) serves as a model system to demonstrate the critical role of electrostatics in EF-hand protein calcium binding.