Real-time-analysis of the calcium-dependent interaction between calmodulin and a synthetic oligopeptide of calcineurin by a surface plasmon resonance biosensor.

The calcium-dependent interaction between calmodulin (CaM) and the synthetic oligopeptide of a predicted CaM-binding region of human calcineurin A-2 was analysed with an automated surface plasmon resonance biosensor, BIAcore. The oligopeptide was immobilized to a biosensor chip via the amino-terminal cysteine residue by a thiol-disulphide exchange method. The biosensor chip was regenerated by an EGTA-containing buffer after each analysis. Kinetics experiments showed that CaM bound with a high affinity to the oligopeptide in a Ca(2+)-dependent manner. The estimated rate constants of association (kass) and dissociation (kdiss) were 2.3 x 10(5) M-1.s-1 and 3.9 x 10(-3)s-1, respectively. The ratio of kdiss/kass, 1.7 x 10(-8) M, was in good agreement with the dissociation constant (Kd) of 2.4 x 10(-8) M determined from the equilibrium phase.