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Figure 14: Standard curve of e -AMP as measured by fluorescence intensity.

63. Figure 14: Standard curve of e -AMP as measured by fluorescence intensity.

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Figure 14: Standard curve of e -AMP as measured by fluorescence intensity.

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  1. 63 Figure 14: Standard curve of e-AMP as measured by fluorescence intensity. e-AMP concentration was determined by absorbance spectroscopy eM265 = 10,000 M-1cm-1 and series of dilutions were prepared from a stock solution in buffer (20 mM Tris, 50 mM NaCl, pH 7.9). The linear fit of this data was determined by linear regression analysis to be excellent with R2 value of 0.9989.

  2. 64 1 2 3 Figure 15: SDS-PAGE analysis of protein preparations. Lane 1: Standards (phosporylase b 97 kDa,serum albumin 66 kDa, ovalbumin 45 kDa, carbonic anhydrase 31 kDa, Trypsin inhibitor 21.5 kDa, and lysozyme 14.4 kDa) lane 2: 10 mg of PE24 lane 3: 12 mg of EF-2

  3. 24530 ± 5 24532.00 100 % 0 mass 20000 21000 22000 23000 24000 25000 26000 27000 28000 29000 30000 65 Da Figure 16 : ESMS analysis of PE24WT. Mass spectra were acquired in the positive ion mode on a Micromass Platform II mass spectrometer equipped with a nanoelectrospray probe by Biological Mass Spectrometry Laboratory at the University of Waterloo.

  4. 66 Figure 17: DSC scans of (A) EF-2 and (B) PE24. The proteins were analyzed in the following buffer systems: PE24 (0.7 mg/ml) in 20 mM TRIS-HCl, 50 mM NaCl, pH 7.0 and EF-2 (1.2 mg/ml) in 20 mM TRIS-HCl, 300 mM KCl, 1 mM EDTA, 5% (v/v) glycerol, pH 8.0. The dotted line in the figure shows the results for the second calorimetric cycle (cooling and re-heating) for the protein.

  5. 68 A B Figure 18: Plot of velocity versus substrate concentartion. (A) As a function of e-NAD+ (EF-2), 20 mM; (PE24), 5 nM; temperature, 25º C. (B) As a function of EF-2. (e-NAD+), 500 mM; (PE24), 20 nM and temperature 25º C. The data are average of triplicate experiments performed at least three times. For more detail refer to methods section.

  6. substrate 69 Table 5: Kinetic Parameters for PE24 ADPRT Activity. The kinetic parameters were determined as described in methods chapter. The values represent the mean  SD from 2 - 6 independent experiments with each experiment consisting of three separate samples.

  7. 70 Figure 19: ADPRT activity of PE24 as a function of pH. ADPRT activity recorded by monitoring the increase in fluorescence. Buffers used:30 mM sodium acetate, pH 2.0-5.0; 30 mM Bis-Tris, pH 6.0-7.0; 30 mM Tris.HCl, pH 7.0-9.0; 30 mM CAPS, pH 10-12. The reaction temperature was 25º C. (e-NAD+), 0-500 mM; (EF-2), 20 mM; and PE24, 20 nM. (A) Kcat versus pH (B) Kcat/KM versus pH.

  8. 72 Figure 20: Effect of temperature on PE24-catalyzed ADPRT activity. Samples containing saturating amounts of EF-2 and various concentrations of e-NAD+ in a range from 50 to 500 mM in 20 mM Tris buffer, pH 7.8, were prepared at room temperature. The ADP-ribosylation activity was measured at various temperatures following a 10-min incubation at each specified temperature. The above data are average of triplicate experiment repeated twice.

  9. 74 Figure 21: ADPRT activity of PE24 as a function of KCl concentration. Assay conditions: buffer; 20 mM Tris, pH 7.9; eNAD+, 200 mM; EF-2, 20 mM; PE24, 20 nM; and temperature 25° C. The activities are expressed relative to that observed with 50 mM KCl.

  10. 75 Figure 22: The effect of KCl concentration on the binding of NAD+ to PE24. KCl (50-600 mM) was included in the assay buffer (20 mM Tris, pH 7.9) and the intrinsic fluorescence quenching of PE24 by NAD+ was measured as described in the Methods section.

  11. S410 S585 S408 G486 T442 S449 S459 S515 S507 77 Figure 23: Ribbon diagram of the C-domain of ETA bound to b-TAD. The positions of amino acids Ser 408, Ser 410, Thr 442, Ser 449, Ser 459, Glu 486, Ser 507, Ser 515, and Ser 585 are indicated. The structure of b-TAD is omitted from the figure even though it is present in the crystal structure.

  12. 82 Table 6: ADPRT activity and NAD+ binding affinity of PE24 and its muteins. The ADPRT activity and NAD+ binding affinity of PE24 and its variants were determined as described in the Methods section. The assays were done in triplicate and each assay was repeated at least twice.

  13. 83 Figure 24: Absorption spectra of AEDANS-PE24WT and AEDANS-S585C-PE24. The spectra are normalized to a value of 1.0. (---) spectrum of AEDANS-S585C-PE24; (—) spectrum of AEDANS-PE24WT treated with IAEDANS.

  14. 84 Table 7: ADPRT activity and NAD+ binding affinity of PE24 and AEDANS derivatives of its variants. The ADPRT activity and NAD+ binding affinity of PE24 variants and its AEDANS derivatives were determined as described in the Methods section. The assays were done in triplicate and each assay was repeated at least twice.

  15. 88 A H H N C H I 2 N O - S PE24 cys muteins S O 3 H H N PE24 cys muteins N S O S O 3 AEDANS derivative of PE24 B Figure 25: Reaction of thiols with iodoacetamide derivatives of two different fluorescent reporters. (A) Reaction of IAEDANS with thiol group of PE24 is shown. The dansyl moiety of IADANS is responsible for its excellent fluorescence properties. (B) Reaction of IAF with thiol group of EF-2. The above two reactions are simple alkylation reactions in which the halide is substituted by the sulfhydryl group forming a stable thioether.

  16. 89 A B Figure 26: Absorption spectra of fluorescently labeled derivatives of PE24 and EF-2. (A) Absorption spectrum of AEDANS derivative of PE24 in 20 mM Tris.HCl, 50 mM NaCl, pH 7.9. (B) Absorption spectrum of fluorescein labeled EF-2 in 20 mM Tris.HCl, 300 mM KCl, pH 7.9. The spectra are normalized to a value of 1.0 at 280 nm.

  17. 90 A B Figure 27: Excitation and emission fluorescence spectra of AEDANS derivative of PE24 and fluorescein derivative of EF-2. (A) Corrected fluorescence excitation (solid line) and emission (dashed line) spectra of 6.0 mM of AEDANS-PE24 in 20 mM Tris.HCl, 50 mM NaCl, pH 7.9. The emission and excitation slits were set to 2 nm each. (B) Corrected fluorescence excitation (solid line) and emission (dashed line) spectra of 3.5 mM of fluorescein-EF-2 in 20 mM Tris.HCl, 100 mM KCl, pH 7.9.

  18. 91 Figure 28: Overlap between the fluorescence spectra of AEDANS-S585C-PE24 and absorption spectra of AF-EF-2. Curve 1 is the emission spectrum of AEDANS-S585C-PE24 at 337 nm (4 nm for both excitation and emission slits); curve 2 is the absorption spectrum of AF-EF-2 with a single cysteine modified.

  19. 92 Figure 29: Detection of fluorescence energy transfer between AEDANS-PE24 and fluorescein-EF-2 upon formation of the specific protein-protein complex. Excitation was at 337 nm with band pass of 4 nm for both excitation and emission. All spectra were recorded in 20 mM Tris.HCl, 50 mM KCl, pH 7.9 buffer at 25 °C. Lanes 1, no EF-2AF; 2, 190 nM EF-2AF; 3, 550 nM EF-2AF; 4, 900 nM EF-2AF; 5, 1.7 mM EF-2AF; 6, 2.4 mM EF-2AF; 7, 3.0 mM EF-2AF.

  20. 93 Figure 30: Titration of AEDANS-S585C-PE24 with AF-EF-2. Titration was performed in 20 mM Tris.HCl, 50 mM KCl, pH 7.9. The quenching of the AEDANS fluorescence (AEDANS-S585C-PE24) was monitored as a consequence of energy transfer by titration of this protein (1.0 mM) with AF-EF-2 (0-3500 nM) as described in Methods. The fluorescence excitation was 337 nm and the emission was 460 nm (4 nm slits) at 25° C.

  21. 95 Figure 31: Binding isotherm for AEDANS-S585C-PE24 to AF-EF-2 in the absence of NAD+. Titration was done in 20 mM Tris, 50 mM KCl, pH 7.9. Final concentration of PE24-AEDANS was 0.7 mM in the assay. Solid line represents the best fit of the data to equation given in the text (page xx).

  22. 97 Table 8: The binding parameters of PE24EF-2 complex in presence and absence of b-TAD (substrate analog). The dissociation constant of PE24.EF-2 complex was determined using non-linear regression analysis. The quenching of the AEDANS fluorescence (AEDANS-S585C-PE24) was determined from the titration of this protein (1.0 mM) with AF-EF-2 (0-5 mM) as described in methods. Errors are the standard deviation from three or more experiments.

  23. 98 Table 9: The binding constant of PE24.EF-2 complex using different derivatives of PE24. The dissociation constant of PE24.EF-2 complex was determined using non-linear regression analysis. The quenching of the AEDANS fluorescence (AEDANS-PE24-muteins) was determined from the titration of each protein (1.0 mM) with AF-EF-2 (0-5 mM) as described in methods. Errors are the standard deviation from three or more experiments.

  24. 101 Figure 32: EF-2 binding of PE24 as a function of KCl concentration. Assay conditions: buffer 20 mM Tris, pH7.9; 1 mM AEDANS-S585C PE24, 4 mM AF-EF-2; and temperature 25° C. The data are average of triplicate experiments repeated three times.

  25. H O N O 1,8-Naphthalimide H H O N O O N H N O 2 N N H 2 GLFD 52 GLFD 09 GLFD 17 H H N H 2 O O H O N O N O O N O GLFD 75 GLFD 85 GLFD 22 103 Figure 33: Structures of inhibitors of ADPRT reaction catalyzed by PE24. All the compounds except Naph were prepared by Guilford Pharmaceuticals.

  26. 104 • Figure 34: Binding isotherm of 1,8-naphthalimide. • Titrations were conducted in 20 mM Tris.HCl, 50 mM NaCl, pH 7.9, at 25C using 1.25 M PE24. The experiments were performed using excitation at 295 nm (4 nm slit width) with fluorescence emission set at 340 nm (4 nm slit width). The data are corrected for the dilution effect.

  27. 106 Table 10: IC50 and KD values for ADPRT inhibitors. IC50 values were determined using the fluorescence-based ADPRT assay as described in methods section. The dissociation binding constants were determined in 20 mM Tris.HCl, 50 mM NaCl, pH 7.9 at 25 °C. The IC50 values were determined for two separate experiments done in triplicates. The KD values were determined for two separate experiments done in duplicates.

  28. 108 5 4 3 2 1 Figure 35: Inhibition of ADPRT activity of PE24 by Naph: Double reciprocal plot. A typical assay in a final volume of 70 ml consisted:20 mM Tris.HCl, pH 7.9, 0-500 M e-NAD+, 20 M EF-2 and 40 nM PE24. The assay was initiated by the addition of enzyme sample which had been preincubated with the inhibitor at 25C for 10 min. The reaction was monitored for a period of 5 min. Inhibitor concentration used: 1, 0 nM; 2, 100 nM; 3, 150 nM; 4, 200 nM; 5, 250 mM.

  29. 110 Table 11: Reversibility of the inhibitory action of Naph. The reversibility of the inhibition of PE24 by Naph was examined by dialysis of the reaction mixture for extended period (4 days) at 4° C. A sample of PE24 (not treated with the inhibitor) subjected to similar procedure served as control. ADPRT activity of PE24 before and after dialysis was measured in 20 mM Tris at 200 M e-NAD+, 20 M EF-2 according to procedure outlined in methods. The data are averages of 2 independent experiments.

  30. 24530.00 100 % 24532.00 100 25534.00 23713.00 26568.00 0 mass 20000 21000 22000 23000 24000 25000 26000 27000 28000 29000 30000 % 25554.00 23700.00 0 mass 20000 21000 22000 23000 24000 25000 26000 27000 28000 29000 30000 111 A B Figure 36. ESMS spectra of PE24. (A) spectrum of PE24 in presence of 1,8-naphthalimide in 1:100 molar ratio. (B) spectrum of PE24 alone.

  31. 112 Figure 37: NMR spectra of PE24 and Naph reaction mixture.

  32. Figure 38:

  33. 117 Figure 39: Structure of e-NAD+. Excitation: 305 nm; emission: 410 nm

  34. Figure 2.5: Slope Replot of Lineweaver-Burk plot for KI determination. • The presented data are average of 5 independent experiments each done in triplicates.

  35. Figure xx: Effect of temperature on binding constant of EF-2.PE-24 complex.

  36. Figure xx: Binding curves of NAD+ binding to AEDANS-T442C-PE24. (A) Schatchard plot. (B) Hill plot.

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