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.