Kinetics of Ca2+ binding to calmodulin and its tryptic fragments studied by 43Ca-NMR.

The kinetics of calcium ion binding to bovine testis calmodulin and its tryptic fragments have been studied by 43Ca-NMR. The same subdivision of the Ca2+-binding sites of calmodulin into two with slow exchange (high affinity) and two with fast exchange (low affinity) observed at low ionic strength is also encountered at high ionic strength. The effect of 0.1 M KCl is to reduce the exchange rate of the fast process from 1150 s-1 to 520 s-1 at 25 degrees C. Studies of the tryptic fragments TR1C and TR2C, comprising the N- or C-terminal half of calmodulin, respectively, clearly identified Ca2+-binding domains I and II as those with fast exchange (low affinity) and domains III and IV as those with slow exchange (high affinity). Activation parameters are reported for calmodulin and TR1C. Correlation times have been measured for ions bound to the fragments. The obtained values, 5 and 6 ns for TR1C and TR2C, respectively, are of the same order as rotational correlation times for the entire fragment molecules, indicating that the calcium ions do not have any mobility with a correlation time in the ns range within the sites.