Dissociation constants of MTX-SNAP-EGFP for SNAP-eDHFR measured by SNAP-based TR-FRET titration assay.

Values are the mean±standard deviation (SD) from 2–6 independent experiments. Receptor concentrations used in each experiment in the absence of NADPH were 0.02, 0.3, 1 and 3 nM for WT, 0.3, 1 and 3 nM for L54I, 1 and 3 nM for L54G and F31V, L54G. Receptor concentrations used in the presence of NADPH were 0.02, 0.03, 0.1 and 0.3 nM for WT, 0.1, 0.3 and 0.5 nM for L54I, 0.1, 0.5 and 30 nM for L54G and F31V, L54G. * indicates that Kd values were calculated with Fmax of the higher affinity samples (see materials and methods).