Figure 4. Extensive self-association of ESAT-6.

(A) BLI long-association experiment showing 50 µM ESAT-6 at pH 4.5 or 7.5 attaching to biosensors coated with either ESAT-6 or CFP-10. (B) Turbidity assay showing 50 µM ESAT-6 or 50 µM CFP-10 at pH 4.5 or 7.5 over time. Absorbance at 350 nm read every 2 min. (C) Lysine-labeled Biotinylated ESAT-6 has an average of 1 biotin per protein attached to the N-terminus or one of the orange highlighted lysine residues. The ESAT-6-LPETG construct amino acid sequence is shown which includes and inserted LPETG motif, in orange. Reaction with biotinylated poly-glycine via sortase-mediated transpeptidation results in the teal highlighted residues being removed and replaced with a biotin molecule. BLI experiments at (D) pH 7.5 and (E) pH 4.5 showing unmodified soluble ESAT-6 interacting with streptavidin biosensor tips loaded with ESAT-6-Lysine-Biotin (red) or ESAT-6-LPETG-Biotin (blue). Curves were normalized by dividing response values by the amount of protein added during the loading step. (F) Summary of apparent kinetic binding constants in (D–E).