Table 3.

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

Reference titrations of SC(1–246)-BODIPY with ProT^QQQ were obtained at two fixed probe concentrations. Competitive binding data were obtained by titrations of fixed concentrations of SC(1–246)-BODIPY probe, and 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.” ND, not determined; SF, stoichiometric factor.

SC(1–246) mutants	SF (m)	KC	ΔFmax/Fo	ΔG	KD from ProTQQQ activation	Vlim	REU complex (predicted)
	mol ProTQQQ/mol SC (1–246)	nm		kcal/mol	nm		REU
I1V2	1.10 ± 0.05	0.7 ± 0.2	0.60 ± 0.01	−12.48	1.5 ± 0.2	1.0 ± 0.01	−130
V1V2	1.17 ± 0.04	0.9 ± 0.3	0.60 ± 0.05	−12.33	2.1 ± 0.5	1.0 ± 0.01	−128
I1A2	1.13 ± 0.02	1.1 ± 0.3	0.57 ± 0.01	−12.25	4.2 ± 0.5	1.8 ± 0.01	−126
L1V2	1.14 ± 0.04	1.3 ± 0.4	0.57 ± 0.004	−12.11	4.1 ± 1.0	1.4 ± 0.1	−117
I1T2	1.01 ± 0.09	5.3 ± 1.7	0.56 ± 0.01	−11.29	5.0 ± 1.4	0.90 ± 0.08	−125
I1W2	1.13 ± 0.12	7.1 ± 2.4	0.59 ± 0.01	−11.11	1.8 ± 0.2	0.92 ± 0.01	481
T1V2	1.02 ± 0.16	11 ± 4	0.56 ± 0.01	−10.88	8 ± 2	1.7 ± 0.1	−124
L1T2	Fixed to 1	19 ± 6	0.60 ± 0.01	−10.54	3.0 ± 0.4	1.4 ± 0.1	−112
L1Q2	Fixed to 1	27 ± 7	0.57 ± 0.01	−10.33	1.2 ± 0.4	1.3 ± 0.1	−108
T1P2	Fixed to 1	61 ± 40	0.57 ± 0.01	−9.84	169 ± 34	0.38 ± 0.03	−57
I1L2	Fixed to 1	64 ± 17	0.57 ± 0.01	−9.82	7 ± 1	1.3 ± 0.1	−119
L1K2	Fixed to 1	76 ± 12	0.60 ± 0.01	−9.71	3.0 ± 0.3	1.6 ± 0.1	−110
V1G2	Fixed to 1	100 ± 27	0.60 ± 0.01	−9.55	3.1 ± 0.4	1.6 ± 0.1	−122
T1A2	Fixed to 1	100 ± 27	0.60 ± 0.01	−9.55	5.2 ± 0.2	1.3 ± 0.1	−120
W1A2	Fixed to 1	117 ± 36	0.60 ± 0.01	−9.45	ND	ND	1,298
M1L2	Fixed to 1	127 ± 38	0.58 ± 0.01	−9.41	16 ± 2	0.90 ± 0.02	−107
M1W2	Fixed to 1	148 ± 27	0.60 ± 0.01	−9.32	8 ± 1	0.25 ± 0.01	493
G1H2	Fixed to 1	153 ± 43	0.61 ± 0.01	−9.29	346 ± 134	0.02 ± 0.003	−115
G1A2	Fixed to 1	166 ± 42	0.60 ± 0.01	−9.25	46 ± 5	0.17 ± 0.01	−118
Q1K2	Fixed to 1	169 ± 49	0.60 ± 0.01	−9.24	59 ± 8	0.20 ± 0.01	−116
A1W2	Fixed to 1	170 ± 60	0.58 ± 0.01	−9.23	44 ± 5	0.14 ± 0.01	487
M1K2	Fixed to 1	174 ± 54	0.58 ± 0.01	−9.22	9 ± 1	1.1 ± 0.1	−111
W1E2	Fixed to 1	191 ± 54	0.60 ± 0.01	−9.17	5,700 ± 4,200	0.13 ± 0.06	1,307
A1K2	Fixed to 1	191 ± 62	0.58 ± 0.01	−9.16	27 ± 2	0.55 ± 0.11	−116
L1P2	Fixed to 1	220 ± 64	0.58 ± 0.01	−9.08	22 ± 3	0.31 ± 0.01	−50
T1D2	Fixed to 1	248 ± 72	0.58 ± 0.01	−9.01	100 ± 18	∼0.2	−115
S1K2	Fixed to 1	251 ± 76	0.58 ± 0.01	−9	44 ± 2	0.16 ± 0.01	−115
Q1L2	Fixed to 1	277 ± 83	0.58 ± 0.01	−8.94	188 ± 21	0.024 ± 0.001	−111
A1S2	Fixed to 1	295 ± 69	0.60 ± 0.01	−8.91	11 ± 0.4	1.0 ± 0.1	−118
R1R2	Fixed to 1	379 ± 88	0.61 ± 0.01	−8.76	26 ± 5	0.1 ± 0.01	−104
G1P2	Fixed to 1	446 ± 81	0.61 ± 0.01	−8.66	64 ± 6	0.020 ± 0.001	−54
K1A2	Fixed to 1	455 ± 80	0.60 ± 0.01	−8.65	69 ± 20	0.010 ± 0.001	−111
G1D2	Fixed to 1	468 ± 80	0.61 ± 0.01	−8.63	76 ± 7	0.020 ± 0.001	−113
A1T2	Fixed to 1	480 ± 95	0.60 ± 0.01	−8.62	5 ± 0.3	1.0 ± 0.1	−118
E1T2	Fixed to 1	486 ± 80	0.60 ± 0.01	−8.61	105 ± 11	0.030 ± 0.001	−113
N1D2	Fixed to 1	493 ± 99	0.60 ± 0.01	−8.6	89 ± 7	0.020 ± 0.001	−113
G1G2	Fixed to 1	503 ± 90	0.61 ± 0.01	−8.59	26 ± 2	0.26 ± 0.01	−115
R1Q2	Fixed to 1	503 ± 94	0.60 ± 0.01	−8.59	35 ± 26	0.004 ± 0.010	−104
M1E2	Fixed to 1	514 ± 96	0.60 ± 0.01	−8.58	56 ± 8	0.060 ± 0.001	−105
E1S2	Fixed to 1	650 ± 120	0.61 ± 0.01	−8.44	205 ± 14	0.010 ± 0.002	−112