J. Phys. Chem. B 2009, 113, 6873–6880
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Molecular Insight into Protein Conformational Transition in Hydrophobic Charge Induction Chromatography: A Molecular Dynamics Simulation Lin Zhang, Guofeng Zhao, and Yan Sun* Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin UniVersity, Tianjin 300072, China ReceiVed: NoVember 5, 2008; ReVised Manuscript ReceiVed: February 25, 2009
Hydrophobic charge induction chromatography (HCIC) is an adsorption chromatography combining hydrophobic interaction in adsorption with electrostatic repulsion in elution. The method has been successfully applied in the separation and purification of antibodies and other proteins. However, little is understood about protein conformational transition and the dynamic process within adsorbent pores. In the present study, a pore model is established to represent the realistic porous adsorbent composed of matrix and immobilized HCIC ligands. Protein adsorption, desorption, and conformational transition in the HCIC pore and its implications to the separation performance are shown by a molecular dynamics simulation of a 46-bead β-barrel coarse-grained model protein in the adsorbent pore. Repeated adjustment of both protein position and orientation is observed before reaching a stable adsorption. Once the protein is adsorbed, there is a dynamic equilibrium between unfolding and refolding. The effect of hydrophobic interaction strength between protein and ligands on adsorption phenomena is then examined. Strong hydrophobic interaction, representing the presence of high-concentration lyotropic salt in mobile phase, can speed up the adsorption but cause protein unfolding more significantly. On the contrary, weak hydrophobic interaction, representing the absence of a lyotropic salt or the presence of a chaotropic agent, can reserve native protein conformation but does not lead to stable adsorption. In the elution, protein unfolding occurs due to simultaneous hydrophobic adsorption and electrostatic repulsion in the opposite directions. When the protein has been desorbed, the conformational transition between unfolded and native protein is still observed due to the long-range nature of electrostatic interaction. The simulation has provided molecular insight into protein conformational transition in the whole HCIC process, and it would be beneficial to the rational design of ligands and parameter optimizations for high-performance HCIC. 1. Introduction Hydrophobic charge induction chromatography (HCIC)1 is an adsorption chromatography combining hydrophobic interaction in adsorption with electrostatic repulsion in elution. The method is based on the pH-dependent behavior of hydrophobic ligands with a weak base group such as pyridyl and imidazolyl.2 Because of the high ligand density, protein adsorption occurs under physiological conditions. Therefore, neither preconcentration nor modification of ionic strength in a feedstock is necessary. Elution is then induced by electrostatic repulsion once the pH in mobile phase is reduced, where both the ligand (weak base group) and the adsorbed protein carry positive charges. Compared with traditional hydrophobic interaction chromatography and ion exchange chromatography, HCIC is advantageous in its salt-independent adsorption, high capacity related to the high ligand density, and easy elution of the captured products. To date, HCIC has been successfully applied in the separation and purification of antibodies2–8 and various other proteins.5,9–11 A corresponding theoretical model has been proposed by Ghose et al.12 to describe the effect of pH and salt concentration on the retention behavior. Various experimental studies have also been performed, including comparison of ligands,10 examination of both salt-independent adsorption and facile elution behavior,2 and verification of separation perfor* Corresponding author. Tel.: +86-22-27404981. Fax: +86-22-27406590. E-mail:
[email protected].
mance on penicillin acylase.11 However, little is known about the dynamics process and protein conformational transition within adsorbent pores due to the lack of powerful microscopic experimental techniques, which restricts the research on both ligand design and process optimization. Thus, in the present study, molecular dynamics (MD) simulation is performed to explore the molecular insight into HCIC. MD simulation13 is a powerful tool with sufficiently small scale in both time and space, and thus it can offer clear microscopic information in a direct manner. It has been widely used to understand protein conformational transition at molecular level resolution,14,15 and is becoming a fundamental technique complementary to the experimental and theoretical studies.16 In this work, a pore model was established to represent the realistic porous adsorbent, including both matrix and immobilized ligands. Protein conformational transition in HCIC pore and its implications to separation performance was investigated by a MD simulation of a 46-bead β-barrel coarsegrained model protein.17 The study was started with a 100 ns MD simulation on the empty pore model to simulate the preequilibration of column. The spatial distribution of ligands was shown. Then, a native protein molecule was randomly placed into the pore and a 100 ns MD trajectory was generated to explore the conformational transition and the dynamic information in adsorption. Hydrophobic interaction strength between the protein and immobilized ligands was adjusted to investigate the effect of salt concentration in mobile phase. Finally, both
10.1021/jp809754k CCC: $40.75 2009 American Chemical Society Published on Web 04/17/2009
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J. Phys. Chem. B, Vol. 113, No. 19, 2009
Zhang et al.
Figure 1. Protein and HCIC adsorbent pore models: (a) native structure of 46 β-barrel protein, (b) ligand model, and (c) HCIC adsorbent pore model. The hydrophobic, hydrophilic, and neutral beads of the protein are drawn in gray, red, and blue, respectively. Beads H1 and H2 of the ligands are drawn in green and HQ is drawn in yellow. The beads in matrix are drawn in purple. The bottom radius of adsorbent pore is 10.89 nm and the height is 19.76 nm. The inset in (c) shows a part of adsorbent pore around protein for a clear view.
TABLE 1: Potential Energy of Model Protein potential
formula
parameter
bond
Vb(rij) ) kb(rij - b0)
angle
Va(θijk) ) kθ(θijk - θ0)2
dihedral
Vφ(φijkl) ) kφ1(1 + cos φ) + kφ2(1 + cos 3φ)
LJ Coulomb
VLJ ) ∑i
