Stabilizing Exposure of Conserved Epitopes by Structure Guided Insertion of Disulfide Bond in HIV-1 Envelope Glycoprotein
Figure 3
Binding affinity analysis of binding of wild-type and disulfide-stabilized SF162 gp120 to sCD4 and mAbs b12 and 17b.
Overlay of binding of varying concentrations of wild-type SF162 gp120 to (A) sCD4, (C) b12 mAb, (E) VRC01 mAb, (G) 17b mAb and (I) 17b mAb (+sCD4). Overlay of binding of varying concentrations of disulfide-stabilized SF162 gp120 (gp120 L1-SS-L2) to (B) sCD4, (D) b12 mAb, (F) VRC01 mAb, (H) 17b mAb and (J) 17b mAb (+sCD4). ∼250–500 RUs of sCD4 or mAbs, b12, VRC01 or 17b, were immobilized directly onto a CM5 sensor chip via amine coupling. Varying concentrations of gp120s, either wild-type of disulfide-stabilized, were then injected at 80 µl/min, at 25°C with HBS-EP buffer as running buffer. The experimental curves were then fitted to a 1∶1 Langmuir binding model using BIAevaluation software 3.2 (BIAcore Inc). The red lines indicate the experimentally derived curves while the black lines indicate curves generated after fitting the experimental data to the 1∶1 Langmuir binding model (BIAevaluation software 3.0). Kon (association rate, in 1/Ms), Koff (dissociation rate, in 1/s) and Kd (dissociation constant, in M) are indicated for each paired (ligand-analyte) interaction, derived by fitting the experimental data to the 1∶1 Langmuir binding model. Chi2 (of ≤1) refers to goodness of fit of the statistical model to observed experimental data.