title: Genetically Directed Production of Recombinant, Isosteric and Nonhydrolysable Ubiquitin Conjugates.
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.
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, Scotland, UK.
authors.forename: Mathew
authors.initials: M
authors.lastname: Stanley
authors.affiliation: MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee, DD1 5EH, Scotland, UK. s.s.virdee@dundee.ac.uk.
authors.forename: Satpal
authors.initials: S
authors.lastname: Virdee
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism*; D057005:Bioengineering / Q000379:methods; D000072017:Deubiquitinating Enzymes; D017021:Methanosarcina barkeri / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D010091:Oximes / Q000737:chemistry; D058105:Polymerization; D014452:Ubiquitins / Q000737:chemistry*
chemical_list: D010091:Oximes; D014452:Ubiquitins; D000072017:Deubiquitinating Enzymes; D000604:Amino Acyl-tRNA Synthetases
keywords: genetic code expansion; isopeptide; oxime; polymerization; synthetic methods; ubiquitylation