Figure 1. Designing disulfide bonds between HflK and HflC to stabilize the closed conformation of the HflK/C membrane assembly.

(A) Positions of cysteine substitutions in HflK (Ala²⁷⁰ and Ala²⁸³) and HflC (Ala²³³ and Ala²⁶⁴). The mutations are designed in the hat region of the structure and allow each HflK to be crosslinked to two neighboring HflC subunits, and vice versa. HflK and HflC are shown in blue and cyan, respectively, in the cartoon representation.

(B) SDS-PAGE analysis of affinity-purified FtsH·HflK/C (right lane) under reducing and non-reducing conditions, with molecular weight standards in the left lane. Under reducing conditions, distinct bands corresponding to HflK and HflC are observed; under oxidizing conditions, these merge into a high-molecular-weight band that does not migrate through the gel, indicating disulfide bond formation under non-reducing conditions.