0 2 4 6 8 10

0

50000

100000

150000

200000

250000

Flu

ores

cenc

e In

tens

ity(A

rbitr

ary

Uni

ts)

[-AMP] (M)

63

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

-AMP concentration was determined by absorbance spectroscopy M

265 = 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 with R2 value of 0.9989.

1 2 3

Figure 15: SDS-PAGE analysis of protein preparations. Lane 1: Standards (phosphorylase b 97 kDa, serum albumin 66

kDa, ovalbumin 45 kDa, carbonic anhydrase 31 kDa, Trypsin inhibitor 21.5 kDa, and lysozyme 14.4 kDa). The gel was stained using Coomassie Brilliant Blue. lane 2: 10 g of PE24lane 3: 12 g of EF-2

64

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 the Biological Mass Spectrometry Laboratory at the University of Waterloo.

24532 ± 5

20000 21000 22000 23000 24000 25000 26000 27000 28000 29000 30000mass0

100

%

24532.00

65

Da

20 25 30 35 40 45 500.0008

0.0010

0.0012

0.0014

Tm 310C

Temperature 0C

Cp

cal/0 C

40 60 800.0000

0.0005

0.0010

0.0015T

m 58.3

0C

Temperature 0C

Cp

cal/0 C

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.

77

0 117 234 351 468 585 702

0

40

80

120

160

v o (p

mo

l/min

)

[NAD+] M

A

B

Figure 18: Plot of velocity versus substrate concentartion. (A) As a function of -NAD+ (EF-2), 20 M; (PE24), 5 nM;

temperature, 25º C. (B) As a function of EF-2. (-NAD+), 500 M; (PE24), 20 nM and temperature 25º C. The data are average of triplicate experiments performed at least three times. The data was fitted to Michaelis Menten equation shown below. The illustrated error is the SD of 9 data set for each point.

79

0 5 10 15 20

0

200

400

600

800

1000

V0

(pm

ol/m

in)

[EF-2] (M)

VV S

K SoM

max [ ]

[ ]

Parameter NAD+ eEF-2

KM (M) 275 52 8.0 1.8

Vmax (pmol.min-1) 234 30 258 24

kcat (min-1) 675 85 734 67

kcat/KM (M-1.min-1) 2.5 106 92.8 106

substrate

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.

Table 5: Kinetic Parameters for PE24 ADPRT Activity.

70

2 4 6 8 10 12

0

100

200

300

400

500

600

700A.

k cat

pH

0 2 4 6 8 10 12

0

1

2

3

4

5

B.

pH

k cat/k

M (

M-1m

in-1)

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. (-NAD+), 0-500 M; (EF-2), 20 M; and PE24, 20 nM. (A) kcat

versus pH (B) kcat/KM versus pH. The data were fit to the following equation using Microcal Origin 6.1 software.

71

y yA

Weo

x x

wc

2

22

2

b g

5 10 15 20 25 30 35 40 45-5

0

5

10

15

20

25

30

35

40

Vm

ax(p

mo

l/s)

Temperature (0C)

Figure 20: Effect of temperature on PE24-catalyzed ADPRT activity. Samples containing saturating amounts of EF-2 and various concentrations of-NAD+ in a range from 50 to 500 M 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 averages of two separate experiments performed in triplicate. Linear regression analysis of the data was used to generate the fitted lines. Note that the data point at 30° C was used as the breakpoint for regression analysis.

82

0 100 200 300 400 500 600 700 800

0.0

0.2

0.4

0.6

0.8

1.0

AD

PR

T a

ctiv

ity

[KCl] (mM)

Figure 21: ADPRT activity of PE24 as a function of KCl concentration.

Assay conditions: buffer; 20 mM Tris, pH 7.9; NAD+, 200 M; EF-2, 20 M; PE24, 20 nM; and temperature 25° C. The activities are expressed relative to that observed with 50 mM KCl. The data were fitted to the following equation using Microcal Origin 6.1 software. The illustrated error for each data point is the SD calculated for triplicate experiments performed at least 3 times.

84

y y A eo

x x

to

11

b g

0 100 200 300 400 500 600

20

30

40

50

60

70

Kd

[N

AD

+ ] (M

)

[KCl] mM

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 to calculate the dissociation constant for NAD-PE24 interaction. The data was fitted to the following equation using the Microcal Origin 6.1 software.

76

y y A eo

x x

to

11

b g

Figure 23: Ribbon diagram of the C-domain of ETA bound to -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 -TAD is omitted from the figure even though it is present in the crystal structure. The figure was generated using Web Lab Pro 3.7 software and the coordinates deposited to Protein Data Bank (PDB entry 1AER).

78

E486

S585

S515

S507S459

S449

S408

S410

T442

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.

Protein Preparation ADPRT activity (min-1) Kd (M)

WT PE24 675 85 55 6

S408C PE24 630 108 59 3

S410C PE24 184 31 50 6

T442C PE24 132 33 35 3

S449C PE24 234 42 129 7

S459C PE24 310 113 55 10

S486C PE24 930 127 57 8

S507C PE24 1256 61 63 2

S515C PE24 307 78 48 5

S585C PE24 731 145 61 4

Table 6: ADPRT activity and NAD+ binding affinity of PE24 and its muteins.

79

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.

260 280 300 320 340 360 380 400

0.0

0.2

0.4

0.6

0.8

1.0

1.2

No

rmal

ized

Ab

sorb

ance

Wavelength (nm)

84

Table 7: ADPRT activity and NAD+ binding affinity of PE24 and AEDANS derivatives of its variants.

Protein Preparation ADPRT activity (min-1) Kd (M)

WT PE24 675 85 55 6

S408C-AEDANS 435 43 44 10

S410C-AEDANS 367 65 51 11

T442C-AEDANS 8 1 5.3 x 103 0.3

S449C-AEDANS 43 7 546 14

S459C-AEDANS 542 66 36 4

S486C-AEDANS 56 5 115 4

S507C-AEDANS 268 30 30 4

S515C-AEDANS 396 73 87 15

S585C-AEDANS 192 61 30 3

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.

85

SO3

NN CH2I

O

H

H

SO3

NN

O

H

HS

PE24 cys muteins-S

PE24 cys muteins

AEDANS derivative of PE24

O OHO

NH

O

CH2I

C O-

O

O OHO

NH

O

C O-

O

S

5-Iodoacetamidofluorescein Fluorescein derivative of EF-2

EF-2-S

EF-2

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 IAEDANS 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.

A

B

86

-

-

250 300 350 400 450 500 550 6000.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

No

rmal

ized

Ab

sorb

ance

Wavelength (nm)

260 280 300 320 340 360 380 400

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

No

rmal

ized

Ab

sorb

ance

Wavelength (nm)

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.

A

B

90

200 300 400 500 600 700

0

10000

20000

30000

40000

50000

Flu

ore

sce

nce

inte

nsi

ty

Wavelength (nm)

200 300 400 500 600 700

0

100000

200000

300000

400000

500000

600000

Flu

ore

sce

nce

Inte

nsi

ty

Wavelength (nm)

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 M 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 M of fluorescein-EF-2 in 20 mM Tris.HCl, 100 mM KCl, pH 7.9.

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.

300 350 400 450 500 550 600 650 700

0.0

0.2

0.4

0.6

0.8

1.0

0.0

0.2

0.4

0.6

0.8

1.0

2

1

Abs

orba

nce

Flu

ores

cenc

e in

tens

ity (

x 10

5 )

Wavelength (nm)

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. Curves 1, no EF-2AF; 2, 190 nM EF-2AF; 3, 550 nM EF-2AF; 4, 900 nM EF-2AF; 5, 1.7 M EF-2AF; 6, 2.4 M EF-2AF; 7, 3.0M EF-2AF.

300 350 400 450 500 550 600

0

20000

40000

60000

80000

100000

120000

140000

160000

180000

200000

220000

240000

260000

280000

300000

45

67

3

2

1

Flu

ores

cenc

e in

tens

ity

Wavelength (nm)

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 (1.0M) 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. The data were fitted to the following equation using Microcal Origin 6.1 software. The illustrated error for each of the data points is calculated SD from triplicate experiments repeated at least 3 time.

-500 0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500

40000

60000

80000

100000

120000

140000

160000

180000

Flu

ores

cenc

e in

tens

ity

[AF-EF-2] (nM)

94

y y A eo

x x

to

11

b g

0 1000 2000 3000 4000 5000

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Fra

ctio

nal S

atur

atio

n (

F/

Fm

ax)

[EF-2AF] (nM)

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 M in the assay. Solid line represents the best fit of the data to equation given in the text (page 156).

95

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.

experiment Kd (nM) Bmax

(+) -TAD 1500 405 1.6 0.2

(-) -TAD 1920 150 1.5 0.1

Table 8: The binding parameters of PE24EF-2 complex in presence and absence of -TAD (substrate analog).

97

Protein Preparation EF-2 dissociation constant(M)

S408C-AEDANS 1.2 0.3

S410C-AEDANS 0.9 0.1

T442C-AEDANS 1.2 0.1

S449C-AEDANS 0.9 0.1

S459C-AEDANS 0.9 0.1

S486C-AEDANS 0.8 0.2

S507C-AEDANS 1.1 0.2

S515C-AEDANS 1.0 0.1

S585C-AEDANS 1.5 0.4

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.

99

0 100 200 300 400 500 600 700 800 900800

1000

1200

1400

1600

1800

2000

2200

Kd

(nM

)

[KCl] (mM)

100

Figure 32: EF-2 binding of PE24 as a function of KCl concentration.

Assay conditions: buffer 20 mM Tris, pH7.9; 1 M AEDANS-S585C PE24, 4M AF-EF-2; and temperature 25° C. The data are average of triplicate experiments repeated three times.

Figure 33: Structures of inhibitors of ADPRT reaction catalyzed by PE24.

All the compounds except Naph were obtained from Guilford Pharmaceuticals.

N OO

H

1,8-Naphthalimide

GLFD 17 GLFD 52 GLFD 09

N OO

H

NH2

NO

H

N

OH2N

ONH2O OH NO

H

NO

H

NO

OGLFD 85 GLFD 22 GLFD 75

NN

O

H

O

GLFD 51

NO

H

O

GLFD 50

NO

H

GLFD 60

NH2O

OO

GLFD 46GLFD 00

O

O

H2N

GLFD 79

ON

H

O N

103

Figure 34: Binding isotherm of 1,8-naphthalimide.

Titrations were conducted in 20 mM Tris.HCl, 50 mM NaCl, pH 7.9, at 25C 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 and fitted to the equation below using the Microcal Origin 6.1 software.

0 1000 2000 3000 4000

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

Bo

un

d (M

)

[1,8-naphthalimide] (nM)

105

yPx

P x

1

2

Table 10: IC50 and KD values for ADPRT inhibitors.

Compound IC50 Binding Constant

KD1 KD2

1,8-naphthalimide 87 12 nM 54 6 nM 1.2 0.1 M

GLFD 00 1.0 0.3 M 474 60 nM

GLFD 09 139 15 M 269 50 M

GLFD 17 1.5 0.2 M 9 2 nM 2.7 0.6 M

GLFD 22 1.0 0.4 M 264 28 nM

GLFD 46 > 100 M 20 2.0 M

GLFD 50 7.0 2 M 26 5 nM 2.0 0.5 M

GLFD 51 > 5 M no binding

GLFD 52 > 20 M 3.0 0.5 nM

GLFD 60 121 15 M 1.3 0.1 M

GLFD 75 12 3 M ND

GLFD 79 484 35 M 34 5 nM 50 15 M

GLFD 85 195 30 nM 135 28 nM

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 and the SD calculated for the obtained IC50 values. The KD values were determined for two separate experiments done in duplicates as outlined in the Methods section.

106

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2

0.0

0.5

1.0

1.5

2.0

1/v

(pm

ol-1

.s)

1/[-NAD+] (M-1) x10-2

Figure 35: Inhibition of ADPRT activity of PE24 by Naph: Double reciprocal plot.

A typical assay in a final volume of 70 l consisted:20 mM Tris.HCl, pH 7.9, 0-500 M -NAD+, 20 M EF-2 and 40 nM PE24. The assay was initiated by the addition of enzyme sample which had been pre-incubated with the inhibitor at 25C 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, 250M.

114

1

3

2

4

5

sample ADPRT activity (pmol.min-1)PE24WT (before dialysis) 588 90PE24WT (after dialysis) 299 36PE24WT + 1,8-naphthalimide (before dialysis) 112 10PE24WT + 1,8-naphthalimide (after dialysis) 226 20

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 -NAD+, 20 M EF-2 according to procedure outlined in methods. The data are averages of 2 independent experiments done in triplicates.

Table 11: Reversibility of the inhibitory action of Naph.

116

20000 21000 22000 23000 24000 25000 26000 27000 28000 29000 30000mass0

100

%

24530.00

23713.00 25534.00 26568.00

20000 21000 22000 23000 24000 25000 26000 27000 28000 29000 30000mass0

100

%

24532.00

23700.00 25554.00

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.

117

118

Figure 37: NMR spectra of NATA and Naph reaction mixture.

The spectra of each of the starting materials in DMSO-d6 was obtained and can be found in Appendix D. The above spectrum shows the NMR signals of the starting materials in a mixture and lack of any new peak corresponding to a covalent interaction between the reagents.

Figure 38: Model of Naph bound to the active site of PE24.

The molecular modeling package Sybyl (version 6.7, Tripos Associate Inc.) on a Silicon Graphics Indigo 2 R4400 XZ workstation was used to generate the model presented above. The full coordinates of the catalytic domain of PE24 in complex with -TAD (PDB entry 1AER) was obtained from the Brookhaven Protein Data Bank. For more details see Methods chapter.

116

O

N

N N

N

HN

OHOH

O

PO O-

O

PO O-

O

O

NH2

N

OHOH

O

+

+

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

118