A designed ubiquitin-binding protein selectively enriches and visualizes unanchored ubiquitin.

While protein ubiquitination has been extensively studied, the roles of unanchored ubiquitin and its chains remain less understood, largely due to a lack of specific, high-affinity tools for their enrichment and visualization. To address this, we used high-throughput affinity maturation and computational protein design to engineer novel proteins that selectively bind unanchored ubiquitin. We first enhanced the binding affinity of the zinc finger domain of histone deacetylase 6, a natural unanchored ubiquitin binder, by including its unstructured amino-terminal loop and introducing the V1091L mutation. Using RFdiffusion, we then designed 11 novel ubiquitin-binding proteins (UBiPs), among which UBiP10 showed specificity for unanchored ubiquitin. This protein enriched unanchored ubiquitin from cells and influenza A virions and served as a probe to determine its cellular levels, supporting a relative decrease upon proteasome inhibition. Imaging studies with UBiP10 further revealed a novel unanchored ubiquitin coat around aggresomes, illustrating the potential of UBiP10 as a unique tool to study unanchored ubiquitin in cellular regulation.