Effects of ligand density on hydrophobic charge induction chromatography: molecular dynamics simulation.

High ligand density is usually required in hydrophobic charge induction chromatography (HCIC) for high adsorption capacity. However, it is not clear to what extent the ligand density alters the adsorption and desorption behaviors, or if this leads to the protein conformational transition within adsorbent pores. In the present study, molecular dynamics simulation is performed to examine the effects of ligand density in HCIC using a 46-bead beta-barrel coarse-grained model protein and a coarse-grained adsorbent pore model established in our earlier work. Four ligand densities (1.474, 1.769, 2.212, and 2.949 micromol/m(2)) are simulated at 298.15 K. The simulations indicate that both the capacity and irreversibility of adsorption increase with ligand density. However, it is found that the fastest adsorption occurs at a ligand density of 2.212 micromol/m(2) rather than at the highest density studied. Analyses of adsorption trajectories, protein-ligand interaction energy, and the free energy map indicate that there is repulsion of protein when unfavorable contacts of the protein and ligands occur. There is an enhanced repulsion at 2.949 micromol/m(2), which increases the energy barrier to the transition region and reduces the opportunities to get stable adsorption, thus leading to the decreased adsorption rate. At 2.212 micromol/m(2), however, the repulsion is mild and the high ligand coverage provides abundant opportunities for the protein to get the fastest adsorption and thus causes the maximum unfolding. In the following simulations, complete and irreversible desorption is observed at all ligand densities, in agreement with the easy pH-induced elution behavior of HCIC observed experimentally. It is found that there is a suitable balance between hydrophobic attraction and electrostatic repulsion at 2.212 micromol/m(2), which leads to the slowest desorption kinetics and causes the maximum unfolding. Moreover, analysis of unfolded protein distribution indicates that unfolding occurs mainly on the ligand surface in both adsorption and desorption. The behaviors have been comprehensively elucidated by molecular and thermodynamic analyses.