Fig. 4.

Experimental determination of the ApxC solution dimer. (A) Locations of engineered cysteine residues (marked with spheres Gly12, blue; Lys160, orange) with respect to two dimeric arrangements (dimer III, Left; dimer I, Right). (B) Cysteine cross-linking of purified wild-type (WT) ApxC or cysteine variant. Protein (50 µg) was treated with or without 100 µM CuCl₂ (X-linker) for 30 min at 25 °C to catalyze cross-linking of proximal cysteines before blocking free cysteines with 5 mM N-ethylmaleimide (NEM). Preblock reactions were treated with NEM and EDTA before addition of cross-linker. (C) Locations of Gly12, Ala15, and Ala19 within the N-terminal α-helix at the dimer III interface. (D) Bacterial two-hybrid assay of homodimerization of WT ApxC or indicated variants. Interaction was assessed qualitatively by blue coloration and quantitatively by β-galactosidase activity (Right), expressed as a percentage of the wild type ± SD. (E) Location of an engineered C-terminal deletion (Δ162–172) in context of dimer I. (F) Bacterial two-hybrid assay assessing homodimerization of ApxC (Δ162–172), details as for D.