Synthesizing Designer Ub Conjugates

The inability to use standard recombinant DNA technologies to genetically encode the isopeptide bond linking the Ub C-terminus to substrate lysine residues presents challenges in building defined Ub conjugates. To overcome this problem, linkage-specific conjugating enzymes are often used. Yet, this methodology is limited to the formation of single linkage chains. To gain access to other chain types several groups have developed strategies based on semi-synthesis and total synthesis. While these methods enable production of previously inaccessible Ub oligomers, several practical challenges preclude their widespread use. We sought to devise an alternative strategy—one that utilizes standard recombinant proteins and minimal synthetic manipulations. 

We developed a new method to synthesize a diverse array of Ub chains by exploiting free-radical thiol-ene chemistry. By employing thiol-ene coupling our lab achieved the first syntheses of well-defined branched conjugates. We have also expanded the methodology to generate linear heterotypic chains and homotypic chains, ranging in size between two and seven subunits. Key to the utility of thiol-ene-derived chains is their ability to function as surrogates of native oligomers, as evidenced by similar conformational ensembles and reactivity profiles toward DUBs. Using the transpeptidase sortase, we have also been able to modify individual subunits of homotypic and heterotypic chains with distinct fluorophores. This methodology is useful for dissecting linkage specificities and understanding how DUBs process different chain types. We are currently developing methods to site-specifically anchor chains to any protein of interest.

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