Fig. 7. (A) The relative affinities of rClfA_221–559w.t. (closed circles), rClfA_221–559Y₂₅₆→A (open squares) and rClfA_221–559Y₃₃₈→A (open diamonds) for Fg were examined in an inhibition ELISA-type assay (Smith et al., 1993). Biotin-labeled rClfA_221–559 was incubated with the indicated increasing concentration of unlabeled ClfA proteins in Fg-coated microtiter wells. The amount of labeled ClfA protein bound to the immobilized Fg was then quantitated. The K_d of each protein was calculated by determining the affinity of the proteins for a fluorescein-labeled 17-amino-acid-long synthetic peptide representing the C-terminus of the Fg γ-chain using fluorescence polarization. (B) The relative affinities of rClfA_221–559A₂₅₄→S (upright closed triangles), rClfA_221–559I₃₈₇→S (inverted closed triangles), rClfA_221–559 K₃₈₉→A (×), rClfA_221–559P₃₃₆→A (+), rClfA_221–559P₃₃₆→S (open upright triangles) for Fg were examined in the same way as above. K_d was also calculated as indicated.