Table 3.

Affinities and ΔΔG of alanine-substituted BoNT/A H_C mutants.

BoNT/A HC mutant	HuC25 FABKD (M)	ΔΔGmut-wt kcal/mol
wt	1.07 ×10−10	-
N918A	6.70 ×10−10	0.9
L919A	7.44 ×10−10	1
E920A	1.31 ×10−9	1.3
F953A	2.15 ×10−8	3
R1061A	7.51 ×10−10	1
D1062A	6.06 ×10−10	0.9
T1063A	6.42 ×10−10	0.9
H1064A	1.35 ×10−5	6.8
Y1066A	4.27 ×10−10	0.7
BoNT/A HC mutant	3D12 FAB KD (M)	ΔΔGmut-wt kcal/mol
Wt	1.75 ×10−10	-
G1129A	1.84 ×10−5	6.8
I1130A	7.35 ×10−9	2.2
R1131A	1.00 ×10−5	6.5

The dissociation equilibrium constant (K_D) for HuC25 (or 3D12) F_AB was calculated for each alanine mutant. The difference in binding free energy (ΔΔG_ala-wt) between the alanine-substituted and wild type (wt) H_C was calculated according to the formula ΔΔG=RTln(K_D,Ala/K_D,_wt) (Materials and Methods). Mutants in bold have ΔΔG_mut-wt ≥1.