Skip to main content
. 2024 May 28;12:RP91930. doi: 10.7554/eLife.91930

Figure 6. An ESAT-6-binding alpaca nanobody.

Figure 6.

(A) ELISA experiment on ESAT-6-coated plates showing nanobody E11rv and an irrelevant isotype control. E11rv displays an EC50 of 17.5 (95% CI 16.0–19.4) µg/mL. (B) BLI experiments with E11rv-coated biosensors tested against pMRLB.7 ESAT-6 (blue) or ESAT-6-LPETG (red) at 10, 3.3, 1, and 0.3 µM. (C) Summary of kinetic binding constants from (B). (D) Schematic diagram of hydrogen-deuterium exchange (HDX) experiment showing apo ESAT-6 or E11rv-bound ESAT-6 incubated in D20, then subjected to digestion and mass spectrometry to measure the levels of deuterium incorporation at different times for different peptides, then mapping this to the protein sequence to identify areas with less or greater deuterium exchange between the apo and bound conditions. (E) HDX data for ESAT-6 and E11rv showing reduced deuterium uptake around residue 50 and increased uptake around residue 75. (F) HDX shifts mapped to the ESAT-6 structure (PDB: 3FAV).