Fig. 3
From: FimH as a scaffold for regulated molecular recognition
Conformational dependence of binding by CDR-6xH variants to Penta-his antibody. (a) Binding of Penta-His to CDR1-6xH variants using ELISA, where KD = 169 ± 16 nM (loose conformation, open squares), 38 ± 4 nM (tight conformation, open circles), 32 ± 3 nM (tight conformation + mab21, filled circles). (b) Binding of Penta-His to CDR2-6xH variants using ELISA, where KD = 7.5 ± 0.4 nM (loose conformation, open squares), 21 ± 1 nM (tight conformation, open circles), 52 ± 3 nM (tight conformation + mab21, filled circles). (c) Binding of Penta-His to CDR3-6xH variants using ELISA, where KD = 2.8 ± 0.3 nM (loose conformation, open squares), 3.6 ± 0.2 nM (tight conformation, open circles), 3.70 ± 0.20 nM (tight conformation + mab21, filled circles). (d) Sample data for binding of CDR2-6xH-L pili to Penta-His using bio-layer interferometry (BLI), with a fit to a two-state (conformation change) reaction model. (e) Sample data for binding of CDR2-6xH-T pili to Penta-His using BLI showing that binding is not concentration-dependent. (f) Binding measured at the end of the association phase during BLI, just before the dissociation phase, for both variants of pili. Binding curves using ELISA (n = 3, mean ± standard deviation) were fitted using GraphPad Prism 7 software using a 1:1 binding model. Error bars in BLI graphs represent the standard deviation of n = 2 experiments on separate days. * indicates p < 0.05 by 1-tailed Student’s t-test