Information embedded in different ubiquitin chains is transduced by proteins with ubiquitin-binding domains (UBDs) and erased by a set of hydrolytic enzymes referred to as deubiquitinases (DUBs). Understanding the selectivity of UBDs and DUBs is necessary for decoding the functions of different ubiquitin chains. Critical to these efforts is the access to chemically defined ubiquitin chains bearing site-specific fluorescent labels. One approach toward constructing such molecules involves peptide ligation by sortase (SrtA), a bacterial transpeptidase responsible for covalently attaching cell surface proteins to the cell wall. Here, we demonstrate the utility of SrtA in modifying individual subunits of ubiquitin chains. Using ubiquitin derivatives in which an N-terminal glycine is unveiled after protease-mediated digestion, we synthesized ubiquitin dimers, trimers, and tetramers with different isopeptide linkages. SrtA was then used in combination with fluorescent depsipeptide substrates to effect the modification of each subunit in a chain. By constructing branched ubiquitin chains with individual subunits tagged with a fluorophore, we provide evidence that the ubiquitin-specific protease USP15 prefers ubiquitin trimers but has little preference for a particular isopeptide linkage. Our results emphasize the importance of subunit-specific labeling of ubiquitin chains when studying how DUBs process these chains.

Covalent attachment of ubiquitin to target proteins is one of the most pervasive post-translational modifications in eukaryotes. Target proteins are often modified with polymeric ubiquitin chains of defined lengths and linkages that may further undergo dynamic changes in composition in response to cellular signals. Biochemical characterization of the enzymes responsible for building and destroying ubiquitin chains is often thwarted by the lack of methods for preparation of the appropriate substrates containing probes for biochemical or biophysical studies. We have discovered that a yeast ubiquitin C-terminal hydrolase (Yuh1) also catalyzes transamidation reactions that can be exploited to prepare site-specifically modified polyubiquitin chains produced by thiol-ene chemistry. We have used this chemoenzymatic approach to prepare dual-functionalized ubiquitin chains containing fluorophore and biotin modifications. These dual-functionalized ubiquitin chains enabled the first real-time assay of ubiquitin chain disassembly by a human deubiquitinase (DUB) enzyme by single molecule fluorescence microscopy. In summary, this work provides a powerful new tool for elucidating the mechanisms of DUBs and other ubiquitin processing enzymes.