Table 2. Ca²⁺association and dissociation rate constants for native and mutant CaMs free in solution or bound to BSCaM_IQ.

Ca²⁺ binding was modeled according to a sequential mechanism with a rapidly equilibrating first step (Scheme 1), so these rate constants correspond with the second Ca²⁺-binding step. Values indicated by an “NA” are not applicable; those indicated by an “ND” are too fast to measure in the stopped-flow fluorometer, which has a dead-time of ~1.4 ms. Association rate constants (k_on) designated by a superscript “A” were calculated using the dissociation rate constants (k_off ) given in this table and previously determined equilibrium constants for the second Ca²⁺ binding step (15). Association rate constants for the C-ter EF hand pair in the CaM-BSCaM_IQ complex were derived from a global analysis of the fluorescence time courses presented in Fig. 5. k_off values designated by a superscript “B” were determined using a 5 µl observation cell, which reduces the stopped-flow dead time to below 1 ms, but unfortunately cannot be used with solutions containing BSCaM_IQ due to their higher viscosity. k_off values for the CaM-BSCaM_IQ complex given in parenthesis were derived from a global analysis the fluorescence time courses presented in Fig. 4. All other k_off values were derived from quin-2 fluorescence data (Fig. 3).

	N-ter EF hand pair		C-ter EF hand pair
CaM	kon (M−1s−1)	koff (s−1)	kon (M−1s−1)	koff (s−1)
NC	3.1±1.2×108 A	1152±117B	3.4±2.4×107 A	10.3±0.7
NxC	NA	NA	5.8±3.2×107 A	10.8±0.6
NCx	3.4±1.5×108 A	1298±135B	NA	NA
CaM-BSCaMIQ	kon (M−1s−1)	koff (s−1)	kon (M−1s−1)	koff(s−1)
NC	3.0±1.2×108 A	473.4±37.9 (528.3±3.3)	3.2±0.1×106	11.7±0.5 (13.5±0.1)
NxC	NA	NA	3.1±0.1×106	11.8±0.5
NCx	ND	ND	NA	NA