Individual ligand Contribution to Increased Binding with
Increasing Ligand Density
We next examined the structural conformation adopted by the
agarose-ligand complex to determine the compensatory binding
conformations adopted by both the protein and the ligand to achieve the
desired interaction. Table 2 indicates that as the number of ligands per
agarose increases, the contribution of each individual ligand to the
overall binding score decreases. This is because contributions from the
backbone of the ligand are sterically hindered from binding to the
antibody and increasing the interaction energy. At the highest ligand
density (six ligands per agarose), not all the ligands contact the
protein equally or at all. In addition, the ligands that made contact
differed in their binding score due to differences in the protein
interaction site. Thus, once a strong interaction is made by one ligand,
the remaining ligands need to interact with proximal sites and hence
limit optimal interaction for the remaining ligands. For the low ligand
density agarose, the docking can adjust to incorporate the ligand
backbone or agarose in the final binding conformation and affinity.
Figure 4A, shows the binding site in the context of the overall antibody
molecule. As the number of ligands attached to the agarose molecule
increases, the contribution of the agarose and ligand backbone to the
total binding affinity decreases (Figure 2B). In doing so, the ligand
backbone cannot contribute to the total and individual ligand binding
affinities. Further, beyond two ligand per agarose, there is a
sub-optimal utilization of the attached ligand. This means that in
making optimal contacts to the protein one side of the ligand complex is
directed away from the protein and cannot make contact, thus excluding
one ligand each time.