Table 4.

ProT^QQQ binding and activation by SC(1–246) N-terminal triple mutants

Reference titrations of SC(1–246)-BODIPY with ProT^QQQ were obtained at two fixed probe concentrations. The competitive binding data were obtained by titrations of fixed concentrations of SC(1–246)-BODIPY probe, and triple mutant SC(1–246) as competitor, with ProT^QQQ. Data were fit simultaneously by the cubic equation to obtain the dissociation constant for ProT^QQQ and SC(1–246)-BODIPY (K_O, probe) and mutant SC(1–246) (K_C, competitor); the stoichiometric factor for SC(1–246)-BODIPY (n) and mutant SC(1–246) (m); and the maximum fluorescence intensity (ΔF_max/F_o). Experimental error represents ± 2 S.D. Competitive equilibrium binding studies and data analysis were performed as described under “Experimental procedures.”

SC(1–246) mutants	KC	KD from ProTQQQ activation	Vlim	ΔFmax/Fo	ΔG
	nm	nm			kcal/mol
I1I2V3	4 ± 3	3 ± 1	0.80 ± 0.03	0.57 ± 0.01	−11.51
R1H2W3	35 ± 23	124 ± 48	0.31 ± 0.03	0.58 ± 0.01	−10.16
F1L2Q3	39 ± 25	> 500	0.06 ± 0.05	0.58 ± 0.01	−10.11
E1L2K3	42 ± 27	309 ± 54	0.26 ± 0.01	0.58 ± 0.01	−10.07
E1S2W3	48 ± 32	28 ± 42	0.03 ± 0.01	0.58 ± 0.01	−9.98
G1G2G3	48 ± 33	29 ± 20	0.11 ± 0.01	0.58 ± 0.01	−9.98
D1D2Y3	55 ± 36	545 ± 145	0.19 ± 0.03	0.58 ± 0.01	−9.90