KINETIC CONTROL OF CA(II) SIGNALING - TUNING THE ION DISSOCIATION RATES OF EF-HAND CA(II) BINDING-SITES

EF-hand Ca(II) binding sites share a conserved architecture and are pr evalent in Ca(II) signaling pathways. The ion binding kinetics of thes e sites are carefully tuned to provide the physiologically appropriate activation and inactivation time scales. Here we examine kinetic tuni ng by the side chain at the ninth position of the EF-loop. A model is proposed in which both the size and charge of the side chain contribut e to kinetic tuning. To test this model, the ninth loop position of th e EF-hand-like site in the Escherichia coli D-galactose binding protei n has been engineered and the Tb(III) dissociation kinetics of the res ulting sites have been analyzed. Substitutions at this position are ob served to generate up to 10(4)-fold changes in Tb(III) dissociation ra tes, with little effect on Tb(III) affinity. Furthermore, the observed pattern of rate changes confirm the model's predictions; long side ch ains at the ninth loop position yield slow dissociation kinetics as pr edicted for a steric block, whereas acidic side chains yield slow diss ociation kinetics as expected for an electrostatic barrier.