Fig. 2.
Functional analysis of a disulfide cross-linked ClpB hexamer. (A) Position of engineered disulfide pairs, which were introduced into full-length ClpB. Only the D2 ring is shown. Mutation sites are depicted as spheres, and cysteine pairs are colored in different hues: ClpBR576C/A821C in pink and magenta, ClpBL581C/R776C in light and dark green, and ClpBN746C/R810C in light and dark brown. Neighboring subunits are shown in different gray shades for clarity. Bound nucleotide is depicted as stick model. (B) ClpBR576C/A821C, ClpBL581C/R776C, and ClpBN746C/R810C form high molecular weight, cross-linked oligomers in the presence of ATP after 10 min of cross-linking reaction. In contrast, ClpB, ClpBR576C, and ClpBL581C/R726C that feature a mismatched cysteine pair do not. An asterisk marks the product when performing the cross-linking reaction on ice. (C) Time course of catalyzed cross-linking reaction of ClpBR576C/A821C in the presence of ATP. M, marker; C, disulfide cross-linked ClpBR576C/A821C hexamer after 20 min; G, glutaraldehyde crosslinked ClpBE271A/E668A hexamer. (D) Size-exclusion chromatograms of wild-type (wt) ClpB and crosslinked (xl) ClpBR576C/A821C. (E–G) ATPase (E) and coupled chaperone activities (F, G) of cross-linked ClpBR576C/A821C relative to wild-type (wt) ClpB, non-crosslinked ClpBR576C/A821C, and ClpBR576C. Error bars represent standard deviations of three independent experiments. (H) Coupled chaperone activities of cross-linked ClpBR576C/A821C and cysteine-free ClpB variants featuring the L460A or L396A mutation, or both. Error bars represent standard deviations of three independent experiments.