The FXII KNG domain and protein conformation. (A) FXII-WT (400 nM) was incubated with vehicle (left panel) or an equimolar concentration of IgG 5A12 (right panel). At indicated times, samples were removed into reducing SDS sample buffer, size fractionated by SDS-PAGE, and transferred to nitrocellulose membranes. Immunoblotting was with an HRP-conjugated anti-FXII IgG. Positions of molecular mass standards are shown on the left. Positions of standards for the Z band, and the HC, and LC of FXIIa are indicated at the right of each image. (B) FXII-WT, ΔFXII, FXII-Ala³², FXII-Lys²⁵³, and FXII-Ala³⁴⁶ (400 nM) were incubated overnight (O/N) with an equimolar concentration of IgG 5A12. FXIIa generation was assessed by chromogenic substrate cleavage. (C) FXII-WT (WT), FXIIa-WT (XIIa), FXII-Lys²⁵³, and FXII-Ala³⁴⁶ (200 ng) were size fractionated by SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with IgG 5A12 (left and middle) or polyclonal anti-FXII IgG (right). (D) One hundred nanomolar FXII-WT (WT), ΔFXII, or FXII-WT mixed with 100 nM IgG 5A12 (WT + 5A12) was incubated with PKa (12.5 nM) at 37°C. At indicated times, aliquots were removed and tested for FXIIa generation by chromogenic assay. (E) smFRET efficiency histograms of FXII S544A in the absence (left panel) or presence (right panel) of 100 mM εACA. Representative graphs are shown. Each experiment was done in triplicate. Gaussian distribution is indicated by red dashed lines. The number of each population is indicated at the left of each image.