Figure 4.

ΔΔG of apparent K_D fold change of alanine-scanning mutants. Apparent K_D [nM] of hTKH2 and related mutants were calculated as described in methods section. Then, the fold change for each glycan was calculated as apparent K_D of mutant Ab divided by the apparent K_D of the original Ab (relative apparent K_D). The relative binding free energy (ΔΔG in kcal/mol) was then calculated [ΔΔG = RTln (apparent K_D/reference apparent K_D)]. ΔΔG serves to quantify the effect of the specific amino acid residue mutation to alanine on Ab reactivity: ΔΔG ≅ 0, ΔΔG > 0, ΔΔG < 0, respectively suggest that the mutation has minimal effect, destabilizing effect, or stabilizing effect on Ab binding (9Ac denotes 9-O-acetylation on the sialic acids).