Myriad homotypic and heterotypic polymeric ubiquitin chains have been observed in vivo, and different linkages and lengths are associated with distinct cellular pathways.  To understand the function of a select group of polymers, e.g., those constructed through K11, K48, and K63-linkages, enzymatic synthesis is often employed.  Subsequently, these products are used as probes for studying the selectivities of proteins containing ubiquitin binding domains as well as deubiquitylating enzymes.  However, there are limitations in the types of polymers that can be constructed with these methods.  For instance, it has remained a formidable challenge to construct homotypic polymers comprised of K6, K27, K29, and K33-linkages.  Moreover, well-defined heterotypic polymers containing mixed linkages or branched chains have been unattainable, thereby restricting our understanding to a few types of polymers.  We have developed a method that is amenable to the construction of a diverse range of homotypic and heterotypic ubiquitin polymers. The method, termed ubiquitin thiol-ene coupling (UTEC), relies on the formation of an Ne-Gly-L-homothiaLys isopeptide bond, which we have shown to be hydrolyzed by linkage specific deubiquitylating enzymes to a similar extent as native Ne-Gly-L-Lys linkages.  Using a single ubiquitin variant harboring a specific lysine-to-cysteine mutation and an alkene appended to Gly75 or Gly76, UTEC furnishes homotypic polymers of varying length in quantities sufficient for biochemical and biophysical studies.  Heterotypic polymers are obtained by mixing and matching polymers comprised of different linkages.  UTEC also allows for the production of branched chains.  The simple and straightforward nature of accessing ubiquitin polymers � both hydrolyzable and non-hydrolyzable � through UTEC will aid in unraveling the function of these unique modifications by (i) helping to identify novel proteins bearing ubiquitin-binding domains and (ii) interrogating the selectivity of deubiquitylating enzymes.

Chemical tools for studying ubiquitin signaling

246th National Meeting (2013)

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