Fig. 1. Association of native and mutant CaMs with BSCaM_IQ.

Values for k_obs were derived from mono-exponential fits to time courses for BSCaM_IQ fluorescence measured after addition of CaM. The lowest final CaM concentration produced was in all cases at least 5-fold greater than final BSCaM_IQ concentration. Pseudo-first order conditions were thus maintained so that k_obs = k_on[CaM] + k_off. (A) Replot of k_obs values determined in the absence of Ca²⁺ vs the concentration of CaM. Nominally Ca²⁺-free conditions were produced by including 3 mM BAPTA in all experimental buffers. The k_on and k_off values derived from the linear fit shown are 3.6±0.2×10⁵ M⁻¹-s⁻¹ and 0.9±0.2 s⁻¹. k_on and k_off values derived for Ca²⁺-free N_xCCaM are 3.2±0.1×10⁵ M⁻¹-s⁻¹ and 1.1±0.1 s⁻¹, and values of 2.9±0.1×10⁵ M⁻¹-s⁻¹ and 0.7±0.1 s⁻¹ were derived for Ca²⁺-free NC_xCaM (data not shown). (B) Replot of k_obs values determined in the presence of 250 µM CaCl₂ vs the concentration of NC_xCaM. The k_on and k_off values derived from the linear fit shown are 1.1±0.2×10⁶ M⁻¹-s⁻¹ and 30.9±1.6 s⁻¹. (C) Replot of k_obs values determined in the presence of 250 µM CaCl₂ vs the concentration of native CaM. The k_on and k_off values derived from the linear fit shown are 3.1±0.2×10⁷ M⁻¹-s⁻¹ and 69.4±9.6 s⁻¹. (D) Replot of k_obs values determined in the presence of 250 µM CaCl₂ vs the N_xCCaM concentration. k_on and k_off values of 4.1±0.2×10⁷ M⁻¹-s⁻¹ and 558.8±10.1 s⁻¹ were derived from linear fit shown.