Table 2.

Binding and activation properties of CaM mutants.

I. Ca2+ binding properties of D58N and D95N CaM mutants
Mutant		K1 × 106 (M−1)a	K2 × 106 (M−1)a		Ref.
D58N, N-terminal domainb		1.55 ± 0.08 (3.16 ± 0.13)c	11.5 ± 0.3 (3.98 ± 0.16)		(37)
D95N, C-terminal domainb		1.41 ± 0.03 (2.82 ± 0.14)	3.63 ± 0.11 (28.2 ± 1.4)
II. smMLCK activation and smMLCKp binding properties of M124L and E84K CaM mutants
Mutant	Kact (nM)d	% maximum smMLCK activation	K′Ca2+ (μM)e	KD (nM)f	Ref.
	1.9 (0.8)	81 ± 2 (100 ± 4)			(47)
M124Lg			–	–
	42.7 ± 16.3 (40.4 ± 13.7)	67.5 ± 8.5 (100.0 ± 5.6)			(44)
E84K	5.0 ± 0.4 (1.4 ± 0.7)	~10 (100)	0.69 ± 0.02 (0.17 ± 0.02)	6.8 ± 5.2 (3.5 ± 1.9)	(22)
III. Ca2+ binding properties of E84K CaM mutant
Mutant	K1′ (μM)h	K2′ (μM)h	K3′ (μM)h	K4′ (μM)h	Ref.
E84K	16.6 ± 4.0 (15.0 ± 2.0)	23.4 ± 4.3 (13.0 ± 0.9)	111.3 ± 4.1 (82.4 ± 8.4)	45.4 ± 4.5 (102.0 ± 9.2)	(22)
E84K + RS20	5.1 ± 1.9 (0.4 ± 0.1)	3.2 ± 1.0 (2.5 ± 0.2)	4.6 ± 1.3 (0.8 ± 0.02)	5.8 ± 0.6 (1.1 ± 0.1)

^a K₁ and K₂ are the macroscopic binding constants for the first and second binding events in either N- or C-terminal domain of CaM;

^b N- and C-terminal domains were obtained using proteolytic fragmentation of CaM with trypsin;

^c wild-type CaM data are italicized and given in parenthesis;

^d K_act is the apparent CaM activation constant defined as the concentration of CaM required to achieve half-maximum activity of smMLCK;

^e K′_Ca²⁺ is the apparent Ca²⁺-binding constant for the interaction of Ca²⁺ with CaM-smMLCK complex;

^f K_D is the dissociation constant for the E84K-RS20 complex, where RS20 is a peptide virtually identical to smMLCKp;

^g Differences between the two sets of data reported for M124L are due to different assay conditions and smMLCK substrates;

^h K′_n are macroscopic Ca²⁺ dissociation constants in the absence and presence of RS20 peptide.