Figure 6. ATP hydrolysis by a variant with one binding site does not support function.

(A) Cartoon depiction of the domain structure of mini-ClpX^ΔN. This variant contains two rigid-body units but only one complete ATP-binding site.

(B) Mini-ClpX^ΔN (loading concentration 12 µM) chromatographed at a position expected for a pseudo dimer on a Superose 6 gel-filtration column. The elution position of a single-chain ClpX^ΔN hexamer is shown for reference.

(C) Mini-ClpX^ΔN hydrolyzed ATP at approximately 75% of the basal rate of single-chain ClpX^ΔN when activities were normalized for the number of active sites. Unlike ClpX^ΔN, ATP hydrolysis by mini-ClpX^ΔN was not stimulated by protein substrate (10 µM V15P-titin^I27-ssrA) or repressed by ClpP₁₄ (3 µM). In panels C and E, data are shown as mean ± SD.

(D) The rate of cleavage of a fluorescent decapeptide (15 µM) by ClpP₁₄ (50 nM) in the presence of increasing concentrations of single-chain ClpX^ΔN or mini-ClpX^ΔN was determined in the presence of 1 mM ATPγS.

(E) Unfolding rates of photo-cleaved Kaede-ssrA (5 µM; Glynn et al., 2012) by single-chain ClpX^ΔN or mini-ClpX^ΔN (2 µM) were determined in the presence of 2.5 mM ATP and a regeneration system.