Kinetics of calcium binding to calbindin mutants

Area of Science:

• Biochemistry
• Structural Biology
• Protein Dynamics

Background:

• Calbindin D9k is the smallest known protein with two EF-hand calcium-binding sites.
• Understanding calcium binding kinetics is crucial for protein function.

Purpose of the Study:

• To investigate the kinetics of calcium dissociation from wild-type and mutant bovine calbindin D9k.
• To correlate kinetic data with binding affinity and structural modifications.

Main Methods:

• Stopped-flow fluorescence spectroscopy.
• Utilized the calcium chelator Quin 2.
• Studied five mutants with amino acid substitutions/deletions near the N-terminal calcium-binding site (Site I).

Main Results:

• Mutations in the calcium-binding loop of Site I significantly altered Ca2+ dissociation rates from Site I, with minimal impact on Site II.
• Disrupting a specific hydrogen bond (Tyr-13 to Glu-35) had no observable effect on Ca2+ dissociation.
• Decreased Ca2+-affinity in mutants was mainly attributed to increased Ca2+ dissociation rates (off-rate).
• Second-order rate constants for Ca2+ binding were approximately 10(9) M-1 s-1 at low ionic strength.
• Higher ionic strength (0.1 M KCl) increased Ca2+ dissociation rates by over threefold, suggesting an ionic transition state.

Conclusions:

• Modifications near Site I predominantly influence its calcium dissociation kinetics.
• The off-rate is a key determinant of calcium affinity in calbindin D9k mutants.
• Ionic strength affects the Ca2+ binding transition state.