TABLE 2.

Effect of PEP-19, PEP(28–62), and PEP(39–62) on calcium binding affinity and cooperativity

The macroscopic dissociation constants were derived as described in Material and Methods. All values are the average mean ± S.E. of (n) independent titrations. The square root of the product of macroscopic dissociation constants for each domain is equivalent to the K_Ca50 for binding to each domain. The precision of this value is greater than the individual dissociation constants. The overall decrease in free energy from binding two Ca²⁺ ions to the N- or C-domains (ΔG_tot) was calculated as ΔG_tot = −RT ln(K₁×K₂). The upper limit for the change in free energy due to cooperative Ca²⁺ binding (ΔΔG_c) was calculated as ΔΔG_c = −RT ln(4 × K₂/K₁). Free energy values are in kcal/mole.

	N	C-Domain					N-Domain
		Macroscopic Kd (μM)			ΔGtot	ΔΔGc	Macroscopic Kd (μM)			ΔGtot	ΔΔGc
		Kd1	Kd2	$\sqrt{{{\text{K}}_{\text{d}1}}^{\ast }{\text{K}}_{\text{d}2}}$			Kd3	Kd4	$\sqrt{{{\text{K}}_{\text{d3}}}^{\ast }{\text{K}}_{\text{d4}}}$
CaM	(15)	17 ± 3	0.4 ± 0.1	2.2 ± 0.2	−15.3 ± 0.3	−3.4 ± 0.3	34 ± 4	6.2 ± 1	13.1 ± 1.0	−13.3 ± 0.1	−1.9 ± 0.2
CaM + PEP-19	(6)	4.7 ± 1	2.0 ± 0.2	2.8 ± 0.2	−15.0 ± 0.1	−1.3 ± 0.2	35 ± 6	3.9 ± 0.6	14.0 ± 1.0	−13.2 ± 0.1	−2.3 ± 0.2
CaM + PEP(28–62)	(3)	5.0 ± 1.3	1.7 ± 0.3	2.8 ± 0.1	−15.0 ± 0.1	−1.4 ± 0.3	57 ± 25	4.1 ± 1.4	13.1 ± 0.5	−13.2 ± 0.1	−2.3 ± 0.5
CaM + PEP(39–62)	(3)	1.4 ± 0.3	0.4 ± 0.1	0.7 ± 0.1	−16.6 ± 0.1	−1.6 ± 0.3	36 ± 1	2.6 ± 1.0	8.6 ± 0.7	−13.7 ± 0.1	−2.4 ± 0.4