TABLE 1.

Coupling free energies (ΔΔΔG_c) between residues in NCBD and ACTR

NCBD mutant	ACTR mutant	ΔΔΔGc	Error
		kcal/mol	kcal/mol
I2062V	L1049A	0.09	0.03
I2062V	L1048A	−0.09	0.03
I2062V	I1067V	−0.11	0.02
I2062V	V1077A	−0.12	0.03
I2062V	L1055A	−0.16	0.06
I2062V	I1073V	0.02	0.05
L2067A	L1048A	0.44	0.04
L2067A	L1049A	0.40	0.04
L2067A	L1055A	−0.58	0.07
L2067Aa	I1067Va	−0.32	0.06
L2067A	V1077A	−0.09	0.04
L2067Aa	I1073Va	−0.13	0.10
L2067A	A1061G	−0.17	0.06
L2070A	L1055A	−0.82	0.10
L2070A	L1048A	−0.23	0.09
L2070A	L1049A	0.52	0.06
L2070A	I1067V	−0.33	0.11
L2070Aa	I1073Va	−0.29	0.08
L2070Aa	A1061Ga	−0.94	0.11
L2074A	L1055A	−0.46	0.10
L2074A	I1067V	−0.24	0.09
L2074A	L1048A	−0.15	0.04
L2074A	L1049A	0.49	0.05
L2074A	A1061G	−0.77	0.11
L2074Aa	I1073Va	−0.28	0.08
V2086A	I1073V	−0.13	0.05
V2086A	I1067V	−0.50	0.04
V2086A	V1077A	−0.07	0.04
V2086A	L1055A	−0.32	0.06
V2086Aa	L1048Aa	−0.36	0.06
V2086A	L1049A	−0.08	0.09
V2086A	A1061G	−0.27	0.06
L2087A	I1067V	0.65	0.12
L2087A	V1077A	0.48	0.09
L2087A	L1048A	1.01	0.13
L2087A	A1061G	0.41	0.14
L2087A	I1073V	0.75	0.20
L2087A	L1055A	0.64	0.11
L2096Aa	V1077Aa	−0.17	0.11
L2096Aa	L1055Aa	−0.12	0.26
L2096Aa	L1048Aa	0.01	0.26
L2096Aa	A1061Ga	−0.72	0.21
A2099Ga	L1048Aa	−0.28	0.07
A2099G	L1049A	0.15	0.04
A2099G	L1055A	−0.25	0.05
A2099Ga	I1067Va	−0.07	0.08
A2099Ga	I1073Va	−0.15	0.11
A2099G	A1061G	−0.21	0.07
A2099G	V1077A	−0.12	0.03
I2101V	L1055A	−0.14	0.05
I2101V	V1077A	−0.02	0.03
I2101V	I1067V	−0.10	0.04
I2101V	L1048A	−0.14	0.03
I2101Va	L1049Aa	−0.37	0.06
I2101V	I1073V	−0.42	0.05
I2101V	A1061G	−0.05	0.07
V2109A	V1077A	−0.15	0.04
V2109A	I1073V	−0.05	0.04
V2109A	I1067V	0.09	0.02
V2109A	L1055A	−0.07	0.05
V2109A	L1049A	−0.13	0.04
V2109A	L1048A	0.02	0.03
V2109Aa	L1056Aa	−0.13	0.13

^a k_off^app for the double mutant complex was determined from fitting k_obs versus [ACTR] to the general equation for a reversible association of two molecules (12). Otherwise, k_off^app was determined using displacement experiments as described under “Experimental Procedures.”