In biological systems, nanoparticles (NPs) elicit bioactivity upon interaction with proteins. As a result of post-translational modification, proteins occur in a variety of alternative covalent forms, including structural isomers, which present unique molecular surfaces. We aimed at a detailed description of the recognition of protein isomeric species by NP surfaces. The transient adsorption of isomeric ubiquitin (Ub) dimers by NPs was investigated by solution NMR spectroscopy. Lys63- and Lys48-linked Ub were adsorbed by large anionic NPs with different affinities, whereas the binding strength was similar in the cases of smaller particles. After the incorporation of paramagnetic tags into NPs, the observed site-resolved paramagnetic footprints provided a high-resolution map of the different protein surfaces binding to NPs. The approach described could be extended to further protein isoforms and more specialized NP systems to allow better control of the interactions between NPs and protein targets.

Despite significant advancements in our understanding of ubiquitin-mediated signaling, the influence of the intracellular environment on formation of transient ubiquitin-partner complexes remains poorly explored. In our work, we introduce macromolecular crowding as a first level of complexity towards the imitation of a cellular environment in the study of such interactions. Using NMR spectroscopy, we find that the stereospecific complex of ubiquitin and ubiquitin-associated domain (UBA) is minimally perturbed by the crowding agent Ficoll. However, in addition to the primary canonical recognition patch on ubiquitin, secondary patches are identified, indicating that in cell-mimicking crowded solution, UBA contacts ubiquitin at multiple sites.

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