Although multimodal chromatography offers significant potential for bioseparations, there is a lack of molecular level understanding of the nature of protein binding in these systems. In this study a nanoparticle system is employed that can simulate a chromatographic resin surface while also being amenable to isothermal titration calorimetry (ITC) and solution NMR. ITC and NMR titration experiments are carried out with N-15-labeled ubiquitin to investigate the interactions of ubiquitin with nanoparticles functionalized with two industrially important multimodal ligands. The ITC results suggest that binding to both multimodal ligand surfaces is entropically driven over a range of temperatures and that this is due primarily to the release of surface bound waters. In order to reveal structural details of the interaction process, binding-induced chemical shift changes obtained from the NMR experiments are employed to obtain dissociation constants of individual amino acid residues on the protein surface. The residue level information obtained from NMR is then used to identify a preferred binding face on ubiquitin for interaction to both multimodal ligand surfaces. In addition, electrostatic potential and spatial aggregation propensity maps are used to determine important protein surface property data that are shown to correlate well with the molecular level information obtained from NMR. Importantly, the data demonstrate that the cluster of interacting residues on the protein surface act co-operatively to give rise to multimodal binding affinities several orders of magnitude greater than those obtained previously for interactions with free solution ligands. The use of NMR and ITC to study protein interactions with functionalized nanoparticles offers a new tool for obtaining important molecular and thermodynamic insights into protein affinity in multimodal chromatographic systems.