FIGURE 3.

Rate of dissociation of PEP-19 from native CaM versus Ca²⁺-binding mutants. Dissociation of CaM from PEP-19 was determined using a stopped-flow fluorimeter as described under “Experimental Procedures.” Essentially, donor-labeled CaM (1 μm) bound to acceptor-labeled PEP-19 (10 μm) was rapidly diluted to achieve partial dissociation of the complex, and an increase in fluorescence. Syringe A contained labeled CaM (1 μm) and labeled PEP-19 (10 μm) with 1 mm EGTA or 2 mm CaCl₂. Syringe B contained 1 mm EGTA or 2 mm CaCl₂, but no proteins. Panel A shows the dissociation in the presence of Ca²⁺ (2 mm CaCl₂). Dissociation from native Ca²⁺-CaM is too fast to detect using a stopped flow with a 1.7-ms dead time. Panel B shows dissociation in the absence of Ca²⁺ (1 mm +EGTA).