Fig. 2.

HT-MEK experimental pipeline and TSA-binding measurements for PafA mutant library. (A) Device photo (A, Left) and schematic showing experimental measurements of inhibition. (B) Representative vanadate inhibition curves. Thin lines indicate per-chamber curve fits; thick lines show fit behavior predicted by median K_i for WT (black), H83G (cyan), and A165V mutants (red); for WT, 10 of 64 total measurements are shown for clarity. (C) Comparison between dissociation constants (K_Ds) measured on- and off-chip for vanadate, tungstate, and P_i. Arrows denote K_d limits (see below and Materials and Methods). These limits were not used to calculate the correlation coefficient. (D and E) Volcano plots of vanadate (D) and tungstate (E) association constants (K_a^mutant/K_a^WT) for PafA glycine and valine scanning libraries; marker size indicates the number of replicates and limits are of affinity effects. (F) Comparison of vanadate affinity effects (K_a^mutant/K_a^WT) and catalytic effects (k_cat/K_{M, mutant}/k_cat/K_{M, WT}) for PafA mutants. (G) Comparison of tungstate affinity effects (K_a^mutant/K_a^WT) and catalytic effects. In F and G, the points are colored by the statistical significance of each mutant’s catalytic and affinity effect using statistical tests comparing the effects of each mutant against the WT PafA measurements: mutants that do not differ significantly in catalysis from WT are shown in gray; mutants with significant catalytic defects are colored based on vanadate (F) or tungstate affinity (G) effect (yellow, increase in affinity; purple, decrease in affinity; red, WT-like affinity). The dashed line is the identity line and the solid line the best-fit correlation line. Vertical arrows denote upper and lower K_a limits, affinities too weak to be measured using the range of inhibitor concentrations used or stronger than measured due to low K_M values, respectively. Left arrows denote upper limits for mutants with catalytic activities below the dynamic range of the assay, and right arrows denote lower limits for mutants with MeP K_M values below the lowest substrate concentration used (Materials and Methods).