Table 2.

Shotgun scanning analysis of hGH

wt aa	(wt/Ala)bp	(wt/Ala)α	ΔΔGmut-wt, kcal/mol
K41	1.81	0.77	0.5
Y42	2.51	1.24	0.4
L45	3.31	1.41	0.5
P48	0.82	0.69	0.1
P61	15.40	0.64	1.9
S62	1.17	1.15	0.0
N63	1.93	1.02	0.4
R64	14.33	0.77	1.7
T67	1.28	0.55	0.5
Q68	2.63	1.00	0.6
Y164	1.48	2.08	−0.2
R167	2.77	0.82	0.7
K168	2.42	1.75	0.2
D171	0.99	0.49	0.4
K172	10.54	0.75	1.6
T175	11.15	0.78	1.6
F176	155.50	1.89	2.6
R178	159.00	2.03	2.6
I179	6.27	0.62	1.4

For each of the 19 scanned positions (wt aa), the wt/Ala ratio was determined for the hGHbp selection, (wt/Ala)_bp, and the antibody selection, (wt/Ala)_α. These data were used to calculate the difference in binding free energy between alanine-substituted and wt hGH for binding to hGHbp (ΔΔG_mut-wt), as described in Materials and Methods. Residues in bold have ΔΔG_mut-wt > 1.0 kcal/mol.