Fig. 1.
Substrate design and synthesis. (A) Preassembled, K48-linked Ub3 chains containing a K48R mutation on the distal ubiquitin were ligated onto a noncleavable linear His6-Ub-GFP fusion protein to produce pure Ub3Ub-GFP. (B) E1, E2, ubiquitin, and ATP were added to His6-Ub-GFP to elongate K48-linked ubiquitin chains of varying lengths on the ubiquitin fused to GFP. These resulting substrates were purified from free ubiquitin chains via Ni-NTA resin and crudely fractionated according to chain length via size-exclusion chromatography to produce pools of “long-”, “medium-”, and “short”-chain substrates (UbLUb-GFP, UbMUb-GFP, and UbSUb-GFP, respectively). (C) To produce branched chains, ubiquitin chains of varying length were enzymatically elongated on a di- or triubiquitin linear fusion protein, Ub-Ub-GFP, or Ub-Ub-Ub-GFP, similar to B. (D) Size-exclusion chromatogram and corresponding SDS/PAGE gel for the purification of substrate described in B.