Ca(2+)- and H(+)-dependent conformational changes of calbindin D(28k)

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• Calbindin D(28k) is an intracellular Ca(2+) binding protein with EF-hand motifs.
• Ca(2+) binding proteins function as sensors or buffers, modulating intracellular Ca(2+) signals.
• Understanding calbindin D(28k) function requires characterizing its structural and functional responses to Ca(2+) and pH.

Purpose of the Study:

• To investigate the Ca(2+)-dependent conformational changes of calbindin D(28k).
• To explore the role of exposed hydrophobic surfaces in calbindin D(28k) interactions.
• To determine the effect of pH on calbindin D(28k) structure and stability.

Main Methods:

• 8-anilinonaphthalene-1-sulfonic acid (ANS) binding assays.
• Hydrophobic column affinity chromatography.
• Optical spectroscopy for Ca(2+)-induced conformational changes.
• Urea-induced unfolding to assess protein stability across pH ranges.

Main Results:

• Calbindin D(28k) possesses exposed hydrophobic surfaces in both Ca(2+)-free and Ca(2+)-loaded states.
• Ca(2+) binding induces a conformational change, though less pronounced than in calmodulin.
• Calbindin D(28k) undergoes rapid, reversible conformational changes with increasing H+ concentration (pH decrease).
• Protein stability increases with H+ concentration, with pH-dependent changes localized to EF-hands 1-3.

Conclusions:

• Calbindin D(28k) likely interacts with cellular components via its exposed hydrophobic surfaces.
• The protein functions as a Ca(2+)-sensor protein, with potential for Ca(2+)- and H(+)-dependent target interactions.
• pH significantly influences calbindin D(28k) conformation and stability, adding another layer to its regulatory mechanisms.