Figure 4

Degradation of the knotted fusion protein ThiS-YbeA. (A) Modelled structure of ThiS-YbeA, from Mallam et al.²¹. Left, topology of YbeA. The knotting loop is shown in orange, with the knotted chain in red. Right, modelled structure of ThiS-YbeA. YbeA is coloured as in the topology diagram, ThiS is in green. (B) Degradation of ThiS-YbeA-ssrA. Left, schematic representation of ThiS-YbeA-ssrA: ThiS (green square), YbeA (orange knot) and ssrA-tag (red line), with residue numbering. Right, degradation of ThiS-YbeA-ssrA (10 µM) by ClpXP monitored by SDS-PAGE. (C) Degradation competition experiment with equimolar amounts of PR65/A-ssrA and ThiS-YbeA-ssrA (5 µM each), monitored by SDS-PAGE and (D) subsequent densitometry. The reactions contained 0.5 µM ClpXΔN₆, 1 µM ClpP₁₄ and 4 mM ATP with regeneration system. Times in minutes indicated below the gel. Arrows indicate position of protein species as confirmed by gel band mass spectrometry analysis: PR65/A-ssrA (black circles), full-length ThiS-YbeA-ssrA (pink triangles), degradation intermediate I₁ (ThiS + 37 residues, blue inverted triangles), degradation intermediate I₂ (ThiS, green squares). In theory, the total amount of ThiS-YbeA-ssrA, I₁ and I₂ should equal 1, but it is greater than 1. We think that this discrepancy lies in the fact that the band for the full-length ThiS-YbeA-ssrA overlaps with the band in the gel from the ATP regeneration system, thus leading to some inaccuracies in the exact quantities of the species present. As MS data clearly show I₁ and I₂ come from the parent ThiS-YbeA-ssrA construct, we are not too concerned about this small difference.