FIGURE 7.
Substrate binding by PutP variants. A, equilibrium dialysis-based binding of [3H]proline (0.5 Ci/mmol) by purified PutP. B–E, SPA-based saturation binding of [3H]proline (1 Ci/mmol) using purified PutP-WT (B), PutP-L398S (C), PutP-C344A (D), PutP-Y248G (E), or PutP-P252C (F). Data in B–F represent the mean ± S.E. of triplicate determinations of representative experiments, and data were subjected to nonlinear regression fitting in GraphPad Prism 5. See text for the dissociation constants (Kd) and proline-to-PutP molar binding stoichiometries. Data in A are from five independent experiments and were subjected to global fitting in GraphPad Prism 5.