GFPssrA was purified as previously described (49). Casein FITC from bovine milk was obtained from Sigma (C0528) and loaded onto a G25 column (GE Healthcare) to remove free fluorescein (49). TtClpP and ∆TtClpX were purified using native NiNTA (nickel-charged nitrilotriacetic acid) affinity chromatography followed by SEC using a 16/600 S200 200 pg Superdex column and a final SEC buffer containing 100 mM tris (pH 8.5), 100 mM NaCl, and 5% glycerol. U-[2H,15N],Ile-δ1-[13CH3]TtClpP was expressed as described (33). When expressed in D2O, TtClpP was insoluble and the protein was recovered from E. coli inclusion bodies using denaturing conditions. Briefly, cells were harvested and resuspended in denaturing buffer containing 8 M urea followed by sonication and centrifugation to remove cell debris. The resulting supernatant was loaded onto a NiNTA column and washed several times with denaturing buffer. The column was then equilibrated in refolding buffer [100 mM sodium phosphate (NaPi) (pH 8), 5% glycerol, and 100 mM NaCl] followed by elution with elution buffer [100 mM NaPi (pH 8), 5% glycerol, 100 mM NaCl, and 400 mM imidazole]. The eluted fractions were loaded into a 16/600 S200 200 pg Superdex column, and the fractions corresponding to native ClpP were pulled. EcClpP was purified as described in (50). FtsZ was a gift of T. Hosek and was purified as previously described (50).

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