| OCR Text |
Show 23 PHENIX [8] to a final R/Rfree=0.234/0.290. The IZN36-E560K/PIE12 crystal was isomorphous with the IZN36-WT/PIE12 crystal. The IZN26-E560K/PIE12 model was therefore refined against the data set collected for IZN36-WT and the model was rebuilt accordingly. The IZN36-WT/PIE12 structure was refined using RefMac [10] and PHENIX [8] to a final R/Rfree=0.212/0.266. Structures were checked using the MolProbity program [11]. See Table 2.2 for crystallographic data and refinement statistics. Fluorescence Polarization ! Lyophilized IZN36-WT, -E560K, or -Q577R was dissolved in H2O, centrifuged at 14K rpm for 5 minutes at RT to remove aggregates. Supernatant was saved and concentration was determined using the Edelhoch method [3] with an extinction coefficient of 5690 M-1cm-1. Lyophilized PIE12-TF2 or PIE7TF2 was dissolved in PBS buffer pH 7.4 and treated similarly to IZN36. Concentration of inhibitor was determined using absorbance at 503 nm and an extinction coefficient of 65000 M-1cm-1. This was diluted in PBS buffer pH 7.4 to create a 50 nM inhibitor stock. From the IZN36 stock, a series of two-fold dilutions was made from 2.667 μM to 0.65 nM IZN36 in PBS buffer pH 7.4. 150 μL of each concentration was mixed with 50 μL of 50 nM PIE12-TF2 or PIE7-TF2 for a final concentration of 12.5 nM inhibitor and 2 μM - 0.49 nM IZN36. Samples were equilibrated for one hour at RT protected from light. Using a 384 well flat bottom black polystyrene plate (Corning), 60 μL of each concentration of IZN36WT, -E560K, or -Q577R, with either PIE7-TF2 or PIE12-TF2 was transferred into |
