Table 2.

Shotgun-scanning analysis of hGH_wtand hGH_v

	Wild–type/mutant ratiosb
	hGHR selection				Antibody selection				ΔΔGmut–wtc(kcal/mol)
	hGHwt		hGHv		hGHwt		hGHv		hGHwt		hGHv
Residuea	Wt/Homo	Wt/Ser	Wt/Homo	Wt/Ser	Wt/Homo	Wt/Ser	Wt/Homo	Wt/Ser	Homo	Ser	Homo	Ser
M14W	0.91	4.45	1.21	3.13	1.39	7.33	0.022	0.29	−0.3	−0.3	2.4	1.4
H18D	1.89	4.17	1.64	1.08	2.42	13.7	0.96	0.84	−0.2	−0.7	0.3	0.2
H21N	2.33	1.25	6.23	0.12	1.59	1.36	7.45	0.045	0.2	−0.1	−0.1	0.6
Q22	1.42	0.37	2.88	1.64	0.69	0.43	1.85	1.85	0.4	−0.1	0.3	−0.1
F25	1.02	1.31	2.17	1.31	1.71	1.49	1.50	1.24	−0.3	−0.1	0.2	0.0
D26	0.70	3.63	1.04	1.04	1.22	0.94	0.92	1.92	−0.3	0.8	0.1	−0.4
Q29	1.94	0.75	3.52	0.81	0.92	0.72	5.50	0.80	0.4	0.0	−0.3	0.0
K41I	1.35	1.14	1.14	1.23	0.34	0.91	0.53	1.84	0.8	0.1	0.5	−0.2
Y42H	1.04	0.77	1.21	1.22	0.47	0.90	1.09	2.50	0.5	−0.1	0.1	−0.4
S43	0.84		1.21		1.53		0.94		−0.4		0.0
F44	1.57	2.80	0.92	2.20	2.79	2.03	1.09	0.90	−0.3	0.2	0.2	0.5
L45W	3.52	1.26	1.24	1.71	2.00	1.94	0.70	0.54	0.4	−0.3	0.3	0.7
Q46W	4.88	0.40	0.73	1.67	0.54	0.40	0.46	0.82	1.3	0.0	0.3	0.4
N47	1.42	0.74	1.16	0.78	0.49	1.04	0.86	1.02	0.6	−0.2	0.2	−0.2
P48	1.19	0.61	0.53	0.96	0.84	0.84	0.64	1.28	0.2	−0.2	−0.1	−0.2
T60	1.79	0.25	1.76	0.76	0.79	0.17	1.14	0.43	0.5	0.2	0.3	0.3
P61	4.94	9.55	4.59	6.38	0.69	0.57	0.93	0.98	1.2	1.7	1.0	1.1
S62	1.94		1.47		1.33		2.36		0.2		−0.3
N63	1.50	0.86	2.65	0.31	0.32	0.62	1.09	0.63	0.9	0.2	0.5	−0.4
R64K	1.88	7.55	1.02	3.08	2.58	1.61	0.66	0.30	−0.2	0.9	0.3	1.4
E65	0.42	0.38	0.67	0.61	0.45	0.27	0.42	0.47	0.0	0.2	0.3	0.2
E66	0.59	0.78	0.67	1.63	0.35	0.40	0.39	0.68	0.3	0.4	0.3	0.5
T67	1.50	0.86	3.52	2.20	0.84	0.22	1.47	0.40	0.3	0.8	0.5	1.0
Q68	1.79	0.86	2.65	1.50	1.27	0.42	1.20	0.40	0.2	0.4	0.5	0.8
Y164	1.18	3.13	0.55	5.33	1.00	5.57	0.86	22.3	0.1	−0.3	−0.3	−0.9
R167N	1.32	1.38	9.00	0.38	1.31	0.75	6.15	0.19	0.0	0.4	0.2	0.4
K168	0.51	2.15	0.55	1.20	0.54	1.94	0.98	7.00	0.0	0.1	−0.3	−1.1
D171S	18.3	1.15	2.17		1.44	2.27	1.33		1.5	−0.4	0.3
K172	0.68	11.0	0.86	23.0	0.46	1.38	1.02	0.82	0.2	1.2	−0.1	2.0
E174S	1.00	4.89	0.94		0.41	4.45	0.82		0.5	0.1	0.1
T175	1.05	4.94	1.64	1.09	0.63	1.09	0.58	0.23	0.3	0.9	0.6	0.9
F176Y	2.25	46.5	0.79	46.0	2.32	3.50	0.52	6.15	0.0	1.5	0.3	1.2
R178	2.37	3.32	1.26	17.8	2.57	0.93	2.58	4.35	0.0	0.8	−0.4	0.8
I179T	0.43	3.23	2.24	3.04	0.56	1.00	2.00	0.71	−0.2	0.7	0.1	0.9
R183	0.69	4.53	9.56	9.33	1.00	1.00	6.15	0.034	−0.2	0.9	0.3	3.3

^aThe 35 residues included in the analysis are listed. The first letter denotes the hGH_wt sequence in the single-letter amino acid code, followed by the position number in the hGH sequence. In positions where the hGH_v sequence differs from that of hGH_wt the letter following the position number denotes the hGH_v sequence (e.g., M14W denotes position 14, which is methionine in hGH_wt and tryptophan in hGH_v).

^b Wild-type/mutant ratios were calculated from ~100 sequences selected for binding to either the hGHR-ECD or an anti-hGH monoclonal antibody. Values significantly different from unity (≥4.0 or ≤0.25) are highlighted in bold.

^cThe wild-type/mutant ratios were used to calculate the difference in binding free energy (ΔΔG_mut–wt) corresponding to each serine or homolog mutation as described in Materials and Methods. Binding energy difference values indicating significant deleterious (≥0.4 kcal/mol) or beneficial (≤0.4 kcal/mol) effects are highlighted in bold.