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.
