Abstract
We describe the genetically directed incorporation of aminooxy functionality into recombinant proteins by using a mutant Methanosarcina barkeri pyrrolysyl‐tRNA synthetase/tRNACUA pair. This allows the general production of nonhydrolysable ubiquitin conjugates of recombinant origin by bioorthogonal oxime ligation. This was exemplified by the preparation of nonhydrolysable versions of diubiquitin, polymeric ubiquitin chains and ubiquitylated SUMO. The conjugates exhibited unrivalled isostery with the native isopeptide bond, as inferred from structural and biophysical characterisation. Furthermore, the conjugates functioned as nanomolar inhibitors of deubiquitylating enzymes and were recognised by linkage‐specific antibodies. This technology should provide a versatile platform for the development of powerful tools for studying deubiquitylating enzymes and for elucidating the cellular roles of diverse polyubiquitin linkages.
Highlights
Post-translational modification of proteins with ubiquitin (Ub) regulates various cellular processes, and defects within this pathway result in numerous pathologies.[1]
Simple synthetic peptides containing an aminooxy functionality in place of the e-amino functionality of a lysine residue can undergo bioorthogonal oxime ligation with Ub carrying a Cterminal aldehyde group.[14]
Incorporating 1 (Scheme 1 A) by genetic code expansion based on the Methanosarcina barkeri (Mb) pyrrolysyl-tRNA synthetase (PylS)/tRNACUA pair[15] would extend this technology to recombinant protein substrates, thereby enabling the production of nonhydrolysable conjugates that have unprecedented isostery with the isopeptide bond (Scheme 1 B, Figure S1)
Summary
Directed production of recombinant, isosteric and non-hydrolyzable ubiquitin conjugates Stanley, Mathew; Virdee, Satpal. Citation for published version (APA): Stanley, M., & Virdee, S. Directed production of recombinant, isosteric and non-hydrolyzable ubiquitin conjugates. Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim
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