Effects of Urea on Selectivity and Protein–Ligand Interactions in Multimodal Cation Exchange Chromatography

Abstract

Nuclear magnetic resonance (NMR) and molecular dynamics (MD) simulations were employed in concert with chromatography to provide insight into the effect of urea on protein-ligand interactions in multimodal (MM) chromatography. Chromatographic experiments with a protein library in ion exchange (IEX) and MM systems indicated that, while urea had a significant effect on protein retention and selectivity for a range of proteins in MM systems, the effects were much less pronounced in IEX. NMR titration experiments carried out with a multimodal ligand, and isotopically enriched human ubiquitin indicated that, while the ligand binding face of ubiquitin remained largely intact in the presence of urea, the strength of binding was decreased. MD simulations were carried out to provide further insight into the effect of urea on MM ligand binding. These results indicated that, while the overall ligand binding face of ubiquitin remained the same, there was a reduction in the occupancy of the MM ligand interaction region along with subtle changes in the residues involved in these interactions. This work demonstrates the effectiveness of urea in enhancing selectivity in MM chromatographic systems and also provides an in-depth analysis of how MM ligand-protein interactions are altered in the presence of this fluid phase modifier.

Hot-Spotting with Thermal Scanning: A Ligand- and Structure-Independent Assessment of Target Ligandability

“…We thus made use of the nonionic chaotropic substance urea to counteract these potential effects and to evaluate if one is able to further increase the capacity of HTTS to detect fragment hits. Urea is known to reduce the unfolding enthalpy and thus the stability of proteins through a combination of changes in solvent structure and dynamic properties as well as a higher degree of solvation of nonpolar amino acid residues. , As urea, despite being a fluid phase modifier, can eventually modulate protein–ligand interactions in a very protein- and region-selective manner, we made use of target-specific reference compounds to define the minimal urea concentration required to achieve a maximal thermal shift increase when compared with that in normal buffer conditions.…”

Identification of preferred multimodal ligand‐binding regions on IgG1 FC using nuclear magnetic resonance and molecular dynamics simulations

“…Yu et al (2015) employed coarse‐grained simulations to investigate the preferred binding orientation of lysozyme on an HCIC surface at different ligand densities under a range of salt concentrations. Our lab has been actively involved in studying the preferred binding regions of small proteins in MM CEX systems by employing protein libraries (Chung et al, 2010), MD simulations (Banerjee et al, 2017; Freed et al, 2011; Parimal et al, 2015, 2017), atomic force microscopy (Srinivasan et al, 2017), and nuclear magnetic resonance (NMR) (Chung et al, 2010; Holstein et al, 2012, 2013; Srinivasan et al, 2014).…”

“…Previous work in our group employed two‐dimensional heteronuclear single quantum correlation (2D‐HSQC) NMR experiments to identify binding sites of single‐mode and MM CEX ligands on a small model protein, ubiquitin, and its mutants (Chung et al, 2010; Holstein et al, 2012). NMR was also employed to evaluate the effects of urea on preferred binding regions in MM systems (Holstein et al, 2013). Furthermore, we have employed MM functionalized gold nanoparticles in solution to identify binding regions on ubiquitin using NMR (Srinivasan et al, 2014).…”

Identification of Preferred Multimodal Ligand Binding Regions on IgG1 FC using Nuclear Magnetic Resonance and Molecular Dynamics Simulations

“…Yu et al employed coarse-grained simulations to investigate the preferred binding orientation of lysozyme on a HCIC surface at different ligand densities under a range of salt concentrations (Yu, Liu, & Zhou, 2015). Our lab has been actively involved in studying the preferred binding regions of small proteins in MM CEX systems by employing protein libraries (Chung, Hou, et al, 2010), MD simulations Freed, Garde, & Cramer, 2011;Parimal, Garde, & Cramer, 2015, 2017, Atomic Force Microscopy (AFM) (Srinivasan et al, 2017) and Nuclear Magnetic Resonance (NMR) Holstein, Chung, et al, 2012;Holstein, Parimal, McCallum, & Cramer, 2013;Srinivasan, Parimal, Lopez, McCallum, & Cramer, 2014).…”

“…Previous work in our group employed 2D heteronuclear single quantum correlation (HSQC) NMR experiments to identify binding sites of single mode and MM CEX ligands on a small model protein, ubiquitin, and its mutants Holstein, Chung, et al, 2012). NMR was also employed to evaluate the effects of urea on preferred binding regions in MM systems (Holstein et al, 2013). Further, we have employed MM functionalized gold nanoparticles in solution to identify binding regions on ubiquitin using NMR (Srinivasan et al, 2014).…”