Table 2.

Coupling energies determined from mutant-cycle analyses

							Fold-change
Construct	Agonist	f	SE	b	SE	E	Observed	Predicted	ΔΔG	n
A96Y+Y127A	Cho	2690	400	3530	850	0.76	16.5	169	1.4	3
A96K+Y127E	Cho	4680	190	5510	480	0.85	18.5	0.118	−3.0	3
A96C+Y127C	Cho	7760	460	793	98	9.79	213	0.589	−3.5	3
I49A+A96L	Cho	1980	290	6360	430	0.31	6.77	18	0.6	5
I49Y+A96L	Cho	8110	1000	925	140	8.77	191	468	0.5	5
I49K+A96D	Cho	3640	15	1230	19	2.97	64.6	65,600	4.1	3
I49C+A96C	ACh	2690	320	3340	410	0.80	0.029	673	5.9	3
I49D+Y127K	ACh	3620	99	8590	1100	0.42	0.015	0.013	−0.1	3
I49Y+Y127I	ACh	3700	270	9460	180	0.39	0.014	0.009	−0.2	4
I49K+Y127D	ACh	321	34	7830	60	0.041	0.001	0.001	−0.0	3
I49C+Y127C	ACh	5570	250	9930	1000	0.56	0.020	0.029	0.2	3
Y93H+A96H	None	203	44	8420	1900	0.024	36,500	48,200	0.1	3
Y93R+A96H	None	124	15	12,200	140	0.010	15,400	8950	−0.32	3
A96N+W149S	None	411	5	9380	800	0.044	66,400	73,300	0.06	3
N95W+Y127F	ACh	12200	1400	2510	22	4.88	0.173	0.132	−0.16	3
I49Y+N95W	ACh	1810	380	4990	590	0.36	0.013	0.021	0.30	5

Column definitions are given in Table 1. Fold-change (observed) is E^mutant-pair/E^wt and fold-change (predicted) is the product of the fold-changes in E measured for each mutant of the pair alone. Rate constants for αY127, αY93, and αW149 mutants can be found in Purohit and Auerbach (21,41). The coupling energy, ΔΔG (kcal/mol), is calculated as described in Materials and Methods. The range-coupling-energy is the difference between the largest and smallest ΔΔG values (kcal/mol): 96-127 (Y/A-C/C) = 4.9; 96-49 (C/C-Y/L) = 5.4; 49-127 (C/C-D/K) = 0.3.