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.
Takano
E
E
Laboratory of Human Tumor Viruses, Kyoto University, Japan.
Hatanaka
M
M
Maki
M
M
D000595:Amino Acid Sequence; D015374:Biosensing Techniques; D019703:Calcineurin; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D011485:Protein Binding
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D010455:Peptides; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D002118:Calcium