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. 2015 May 1;10(5):e0125345. doi: 10.1371/journal.pone.0125345

Fig 1. Assembly of ClpP1 and ClpP2 to the ClpP1P2 double-ring particle.

Fig 1

Analytical gel filtration runs were performed on a Superdex 200 gel filtration column (24 ml). Unless noted otherwise, all runs were performed at room temperature. The assembly state of the proteins is indicated by a cartoon depiction in the topmost graph of either single rings in light grey for ClpP1 and dark grey for ClpP2, or the assembled double-ring particle. Molecular size markers are indicated as arrow tips above the elution profiles in the first graph. A marker for E. coli ClpP (EcClpP) is included as a reference for the double-ring assembly. The concentration of ClpP1 and ClpP2 is always 25 μM protomer. A. ClpP1 and ClpP2 containing the N-terminal propeptide (proClpP1, proClpP2) assemble to double-ring complexes in the absence of the activator. When proClpP1 (dashed light grey) and proClpP2 (dashed dark grey) are incubated together at room temperature overnight they assemble into the double-ring complex (dark brown). SDS-PAGE shows a 1:1 ratio of ClpP1 to ClpP2 in the peak fraction. Incubation of the assembled complex at 4°C for 3 hours leads to disassembly (blue). Reincubation of the complex at room temperature for 3 hours leads to reassembly into the double-ring complex (light brown). B. Analytical gel filtration of individually loaded mature ClpP1 (mClpP1, dashed light grey), mature ClpP2 (mClpP2, dashed dark grey) and Δ15NClpP2 (dashed orange). mClpP2 shows its main elution peak at 10 ml corresponding to a size of around 450 kDa. Elution profiles were also recorded after overnight incubation of mClpP1 and mClpP2 (black), and of mClpP1 and Δ15NClpP2 (orange). C. Elution profiles of in vitro processed mClpP1P2. To produce the mature complex proClpP1 and proClpP2 were incubated in the presence of 1 mM activator overnight at room temperature. The activator was then removed by buffer exchange and the mClpP1P2 complex was incubated at 4°C for 3 hours leading to disassembly of the complex (blue). Reincubation of the complex at room temperature for 3 hours leads to reassembly into the double-ring complex (light brown).