Ifenprodil titration curve for Buffer -9 refolded wild type and mutant proteins. A. Average dose-titration plots showing reduced affinity of ifenprodil to the F176A mutant protein (n = 5) as compared to the wild type protein (n = 5). The mutant protein harboring double mutations D101A/F176A (n = 5) (open circle) showed further reduction in affinity compared to the single mutant, F176A (filled circle). B. Mutation at residue Asp113, which was identified outside the putative ifenprodil binding pocket by our homology model, did not affect the binding affinity significantly (n = 5, P > 0.4). The KD values for different mutant proteins are listed in Table 2. Each data point is the average value obtained from five experiments using proteins obtained from at least two independent batches of bacteria induction. C. Mean-fitted KD values determined for WT protein and proteins containing the respective mutations. Paired Student t-Tests were performed to evaluate the difference between mean of KD(mutant) and KD(WT), * P < 0.05.