Electrostatic Contributions to Protein Retention in Ion-Exchange Chromatography. 1. Cytochrome c Variants

Abstract

Among the factors that modulate protein interactions, several protein structural properties, such as size, shape, and charge distribution, may play significant roles. In this work, we investigate the influence of protein structure on binding in ion-exchange chromatography, in which electrostatic interactions are dominant. Chromatographic experiments show separation of cytochrome c variants with a limited number of sequence differences to be feasible. To probe the molecular basis for this behavior, protein-adsorbent electrostatic interactions were modeled in the context of continuum electrostatics accounting for the full 3D protein structure. Protein retention was modeled by averaging over all protein-adsorbent configurations using the full accessible surface of the protein. The electrostatic interaction free energy distribution shows that configurations in which numerous positive protein charges are close to the cation exchanger functional groups produce the most favorable binding. The calculated binding equilibrium constant, found by averaging over the full 3D configurational space, captures the chromatographic differentiation of closely related cytochrome c variants. To obviate the need for full sampling of protein configurations, calculations of interaction free energies at short protein-adsorbent separation distances or of protein surface potentials were found to yield reasonable semiquantitative descriptions of the retention trends.

Impact of denaturation with urea on recombinant apolipoprotein A‐IMilano ion‐exchange adsorption: Equilibrium uptake behavior and protein mass transfer kinetics

“…The characteristics of these charged surface regions and Table 2 and D e values for 0 M, 2 M, and 6 M urea from Table 3. charge distribution can have a dramatic influence on chromatographic behavior [33][34][35]. Upon denaturation, the protein no longer has a surface in the classic sense, and binding of charged residues to the stationary phase may be modulated by the proximity of oppositely charged residues in the primary structure of the protein.…”

Protein adsorption and transport in polymer-functionalized ion-exchangers

“…Even for conventional media at low coverages, the mechanistic basis for retention, specifically the protein-surface interaction, has not yet reached the level of predictability. Simplistic stoichiometric models [71,72] have largely been supplanted by more mechanistic colloidal ones [70,98–105] that can often capture trends correctly but still lack quantitative predictive capabilities. The stoichiometric models can be applied to sorption on polymer-derivatized media as well, but again much of the essential physics is missing, and 3-D analogs to the colloidal models will require incorporation of several additional features discussed in the remainder of this section.…”

Chromatographic and electrophoretic characterization of protein variants

“…In 1989, Chicz and Regnier [36] worked with several genetically engineered subtilisin variants, with multiple amino acid substitutions as well as with single amino acid substitutions, to describe the role of charged and uncharged amino acid residues in the different retention behavior of these protein isoforms on a strong cation-exchange material (GE Healthcare Mono-S column). Yao and Lenhoff [38,39] investigated the influence of electrostatic effects on the retention behavior within the protein-surface system of different proteins (cytochrome c, lysozyme, subtilisin, fibroblast growth factor) on three conventional cation-exchange materials (Toyopearl SP-650 C, Toyopearl SP-550 C, CM Sepharose FF), and explored how individual residues contribute to protein retention in IEC. In the model used for describing the protein-adsorbent surface interaction, the geometry and charge distribution of the protein were explicitly included [38].…”

“…Yao and Lenhoff [38,39] investigated the influence of electrostatic effects on the retention behavior within the protein-surface system of different proteins (cytochrome c, lysozyme, subtilisin, fibroblast growth factor) on three conventional cation-exchange materials (Toyopearl SP-650 C, Toyopearl SP-550 C, CM Sepharose FF), and explored how individual residues contribute to protein retention in IEC. In the model used for describing the protein-adsorbent surface interaction, the geometry and charge distribution of the protein were explicitly included [38]. Electrostatic modeling was found to explain slight differences in the retention behavior of protein variants with small structural variations.…”