(650a) Fundamental Investigation of Protein-Ligand Interactions in Multimodal Chromatography Using Nuclear Magnetic Resonance Spectroscopy and Isothermal Titration Calorimetry

Recently, the use of multimodal (MM) chromatography, nuclear magnetic resonance (NMR) and molecular dynamics simulations (MD) in our group has revealed the presence of a preferred binding face on the surface of ubiquitin. Although trends in chromatographic retention data were consistent with that of NMR, there is a need to quantify effects of synergy and avidity involved in these interactions. A novel approach with systems of varying ligand density was employed to extract quantitative information about the co-operative effects of proteins binding to self-assembled monolayers (SAM) of MM ligands. Gold nanoparticles (AuNPs) were functionalized with a SAM of MMC ligands and titrations were performed on ¹⁵N labeled ubiquitin. Chemical shift perturbations obtained from the ¹⁵N-¹H HSQC spectra were used to determine the residue specific dissociation constants, which provided quantitative information about variation in strength of binding across the protein surface. In the present study two different MM cation exchange ligand systems, namely the ‘Nuvia cPrime’ like ligand and the ‘Capto MMC’ like ligand were employed to delve into the differences in protein binding at a molecular level. Although a similar binding face was observed on Ubiquitin for both these ligand systems, a quantitative comparison revealed a localized binding region on Ubiquitin for the ‘Capto MMC’ system and a more diffuse binding region for ‘Nuvia cPrime’.

In order to investigate the thermodynamic processes governing these interactions, Isothermal Titration Calorimetry (ITC) was employed. Titrations were performed for these protein-nanoparticle systems to obtain quantitative information regarding the overall binding constant, the stoichiometry and thermodynamic parameters which define the driving forces of binding in these systems. Binding in both ligand systems was found to be primarily entropically driven. Titrations were also carried out at different temperatures to further investigate the relative enthalpic and entropic contributions. Finally, the overall binding constants obtained from ITC were compared to residue specific binding constants obtained from NMR in order to study effects of synergy involved in the binding of these systems.

To investigate effects of co-operativity and avidity involved in these systems, nanoparticles were synthesized with different ligand densities of these MM ligands. NMR and ITC were then employed to characterize protein binding to SAMs of varying ligand density. These molecular level investigations could potentially reveal the importance of ligand density on protein selectivity in multimodal systems which could have a significant impact on the development of more selective bioprocesses.