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. 2017 Jan 9;36(4):425–440. doi: 10.15252/embj.201694314

Figure 5. Ubiquitylation‐independent oligomerization of the Rsp5 G747E mutant.

Figure 5

  • A
    Model showing stabilizing hydrogen bonds within the G747 turn‐like structure, as well as hydrogen bonds between the loop (purple) and the α1 (pink) residues in the Rsp5‐HECT structure (3OLM).
  • B
    Size‐exclusion chromatography profile of Rsp5232–809 WT or Rsp5232–809 G747E, run on a Superdex 200 16/60 column.
  • C
    Migration of 6.6 μM and 20 μM of Rsp5232–809 WT or Rsp5232–809 G747E in a native polyacrylamide gel.
  • D
    AUC‐SE analysis in terms of non‐interacting ideal solutes for Rsp5232‐809 WT or Rsp5232‐809 G747E fused to His6‐MBP at 8,000 and 12,000 rpm (see also Table EV1).
  • E, F
    (E) Representative gel and (F) quantification (mean and standard deviation from three replicates) of in vitro ubiquitylation of Rvs167 by Rsp5232–809 WT or Rsp5232–809 G747E, in the presence of fluorescein‐labelled ubiquitin. Reaction set‐up and detection were performed as described in Fig 2C.