Recently, the use of multimodal (MM) chromatography, nuclear magnetic resonance (NMR) and molecular dynamics (MD) simulations in our group has revealed the presence of a preferred binding face on ubiquitin for MM ligand interactions. Although chromatographic retention data and NMR results were consistent, synergy and avidity in these interactions need to be studied quantitatively. 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 were functionalized with SAMs 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 ligand binding strengths to different regions on the protein surface. This approach can be used as a quantitative molecular probe for MM interactions and provide insights into synergistic effects in these systems.

Fundamental investigation of protein-ligand interactions in multimodal chromatography using nuclear magnetic resonance spectroscopy

245th National Meeting (2013)

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