It has long been known that ubiquitin chains modify proteins for distinct cellular function; for instance, Lys48-linked chains target proteins for proteasomal degradation, whereas Lys63- and Met1-linked chains function in cell signaling. These chain types are recognized by ubiquitin-interacting proteins, but how recognition is achieved is not well understood. Ye et al. analyzed different conformations of dual-labeled Lys48-, Lys63- and Met1-linked diubiquitin molecules by single-molecule fluorescence resonance energy transfer (smFRET) or by two-color coincidence detection. Each diubiquitin chain type existed in multiple conformational states in solution. Lys63- and Met1-linked diubiquitin adopted extended 'open' as well as more compact conformations, whereas Lys48-linked diubiquitin adopted predominantly compact conformations. Ubiquitin-interacting proteins, including deubiquitinases (DUBs), were found to bind preexisting conformations of Lys63- and Met1-linked chains, whereas DUBs recognized existing semiopen and open conformations but not the compact conformation. Access to the ubiquitin hydrophobic patches (to which all known DUBs bind) was obstructed in the prevalent compact structure of Lys48-linked diubiquitin, which suggests that DUBs may remodel ubiquitin chains to hydrolyze the isopeptide bond. In fact, mutating the Lys48-diubiquitin interface changed the conformational dynamics and affected DUB activity, which suggests that conformational selection takes place and is functionally important. The authors propose that distinct conformations in different ubiquitin chain types may contribute to the specificity of ubiquitin-interacting proteins and that conformational equilibria in ubiquitin chains provide an additional layer of linkage-dependent regulation within the ubiquitin system. Their research shows that the protein tertiary conformation, as determined by the ubiquitin domains, has a direct impact on the binding and activity of interacting proteins. (Nature 492, 266–270, 2012)