63
250000
Fluorescence Intensity
(Arbitrary Units)
200000
150000
100000
50000
0
0
2
4
6
8
10
[e-AMP] (mM)
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 with R2 value of 0.9989.
64
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 mg of PE24
lane 3: 12 mg of EF-2
65
24532.00
100
24532 ± 5 Da
%
0
20000
21000
22000
23000
24000
25000
26000
27000
28000
29000
mass
30000
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.
77
0
Tm 58.3 C
0.0010
0
Cp cal/ C
0.0015
0.0005
0.0000
40
60
80
0
Temperature C
0.0014
0
Tm 31 C
0
Cp cal/ C
0.0012
0.0010
0.0008
20
25
30
35
40
45
50
0
Temperature 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.
79
A
160
vo (pmol/min)
120
80
40
0
0
117
234
351
468
585
702
+
[NAD ] mM
B
1000
V0 (pmol/min)
800
600
400
200
0
0
5
10
15
20
[EF-2] (mM)
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. The data was
fitted to Michaelis Menten equation shown below. The illustrated
error is the SD of 9 data set for each point.
Vo 
Vmax  [ S ]
K M  [S ]
70
Table 5: Kinetic Parameters for PE24 ADPRT Activity.
substrate
Parameter
NAD+
eEF-2
KM (mM)
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
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.
71
700
A.
600
500
kcat
400
300
200
100
0
2
4
6
8
10
12
pH
5
B.
3
-1
-1
kcat/kM (M min )
4
2
1
0
0
2
4
6
8
10
12
pH
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. (eNAD+), 0-500 mM; (EF-2), 20 mM; 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.
bx  x g
2
2
A
y  yo 
e
W 2
w
c
2
82
40
35
30
Vmax(pmol/s)
25
20
15
10
5
0
-5
5
10
15
20
25
30
35
40
45
0
Temperature ( C)
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 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.
84
1.0
ADPRT activity
0.8
0.6
0.4
0.2
0.0
0
100
200
300
400
500
600
700
800
[KCl] (mM)
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. 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.
b g
y  yo  A1e
 x  xo
t1
76
70
50
+
Kd [NAD ] (mM)
60
40
30
20
0
100
200
300
400
500
600
[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.  x  x
b g
o
y  yo  A1e
t1
78
S585
E486
S515
T442
S410
S408
S449
S459
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. The figure
was generated using Web Lab Pro 3.7 software and the
coordinates deposited to Protein Data Bank (PDB entry
1AER).
S507
79
Table 6: ADPRT activity and NAD+ binding affinity of PE24 and its
muteins.
Protein Preparation
ADPRT activity (min-1)
Kd (mM)
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
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.
84
1.2
Normalized Absorbance
1.0
0.8
0.6
0.4
0.2
0.0
260
280
300
320
340
360
380
400
Wavelength (nm)
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.
85
Table 7: ADPRT activity and NAD+ binding affinity of PE24 and
AEDANS derivatives of its variants.
Protein Preparation
ADPRT activity (min-1)
Kd (mM)
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.
86
A
H
H
N
N
CH2I
O
-
S
PE24 cys muteins
SO3H
H
N
N
S
PE24 cys muteins
O
SO3AEDANS derivative of PE24
B
HO
O
NH
HO
O
O
O
C O-
C O-
O
O
CH2I
O
5-Iodoacetamidofluorescein
NH
-
S
EF-2
S
EF-2
O
Fluorescein derivative of 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.
90
A
1.6
Normalized Absorbance
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
260
280
300
320
340
360
380
400
500
550
600
Wavelength (nm)
B
1.6
Normalized Absorbance
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
250
300
350
400
450
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.
91
A
Fluorescence intensity
50000
40000
30000
20000
10000
0
200
300
400
500
600
700
Wavelength (nm)
B
600000
Fluorescence Intensity
500000
400000
300000
200000
100000
0
200
300
400
500
600
700
Wavelength (nm)
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.
1
1.0
1.0
5
Fluorescence intensity (x 10 )
92
0.8
0.6
0.6
2
0.4
0.4
0.2
0.2
0.0
0.0
300
350
400
450
500
550
600
650
Absorbance
0.8
700
Wavelength (nm)
Figure 28: Overlap between the fluorescence spectra of AEDANSS585C-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.
93
300000
280000
Fluorescence intensity
260000
240000
220000
200000
180000
160000
1
140000
120000
2
100000
3
4
5
6
7
80000
60000
40000
20000
0
300
350
400
450
500
550
600
Wavelength (nm)
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 mM EF-2AF; 6, 2.4 mM EF-2AF; 7, 3.0 mM
EF-2AF.
94
180000
160000
Fluorescence intensity
140000
120000
100000
80000
60000
40000
-500
0
500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500
[AF-EF-2] (nM)
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-S585CPE24) 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. 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.
b g
y  yo  A1e
 x  xo
t1
95
1.2
Fractional Saturation (DF/DFmax)
1.0
0.8
0.6
0.4
0.2
0.0
0
1000
2000
3000
4000
5000
[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 mM in the assay.
Solid line represents the best fit of the data to equation given
in the text (page 156).
97
Table 8: The binding parameters of PE24EF-2 complex in
presence and absence of b-TAD (substrate analog).
experiment
Kd (nM)
Bmax
(+) b-TAD
1500  405
1.6  0.2
(-) b-TAD
1920  150
1.5  0.1
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.
99
Table 9: The binding constant of PE24.EF-2 complex using
different derivatives of PE24.
Protein Preparation
S408C-AEDANS
EF-2 dissociation constant
(mM)
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
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.
100
2200
2000
Kd (nM)
1800
1600
1400
1200
1000
800
0
100
200
300
400
500
600
700
800
900
[KCl] (mM)
Figure 32: EF-2 binding of PE24 as a function of KCl concentration.
Assay conditions: buffer 20 mM Tris, pH7.9; 1 mM AEDANSS585C PE24, 4 mM AF-EF-2; and temperature 25° C. The data
are average of triplicate experiments repeated three times.
103
H
O
N
O
1,8-Naphthalimide
H
O
H
N
O
O
N
H2N
O
N
NH2
GLFD 52
GLFD 17
GLFD 09
H
H
O
NH2
O
OH
O
O
N
N
N
O
O
GLFD 85
N
H
H
H
O
GLFD 75
GLFD 22
O
O
N
N
N
O
O
GLFD 51
GLFD 50
O
GLFD 60
H
O
O
NH2
N
O
H2N
O
GLFD 79
GLFD 00
O
N
O
GLFD 46
Figure 33: Structures of inhibitors of ADPRT reaction catalyzed by
PE24.
All the compounds except Naph were obtained from Guilford
Pharmaceuticals.
105
1.4
1.2
Bound (mM)
1.0
0.8
0.6
0.4
0.2
0.0
0
1000
2000
3000
4000
[1,8-naphthalimide] (nM)
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 mM 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.
y
P1 x
P2  x
106
Table 10: IC50 and KD values for ADPRT inhibitors.
Compound
IC50
Binding Constant
K D1
K D2
54  6 nM
1.2  0.1 mM
1,8-naphthalimide
87  12 nM
GLFD 00
1.0  0.3 mM
474  60 nM
GLFD 09
139  15 mM
269  50 mM
GLFD 17
1.5  0.2 mM
9  2 nM
GLFD 22
1.0  0.4 mM
264  28 nM
GLFD 46
> 100 mM
20  2.0 mM
GLFD 50
7.0  2 mM
26  5 nM
GLFD 51
> 5 mM
no binding
GLFD 52
> 20 mM
3.0  0.5 nM
GLFD 60
121  15 mM
1.3  0.1 mM
GLFD 75
12  3 mM
ND
GLFD 79
484  35 mM
34  5 nM
GLFD 85
195  30 nM
135  28 nM
2.7  0.6 mM
2.0  0.5 mM
50  15 mM
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.
114
2.0
5
1.5
-1
1/v (pmol .s)
4
1.0
3
2
0.5
1
0.0
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2
+
-1
-2
1/[e-NAD ] (mM ) x10
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 mM e-NAD+, 20 mM 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
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.
116
Table 11: Reversibility of the inhibitory action of Naph.
sample
PE24WT (before dialysis)
PE24WT (after dialysis)
PE24WT + 1,8-naphthalimide (before dialysis)
PE24WT + 1,8-naphthalimide (after dialysis)
ADPRT activity (pmol.min-1)
588  90
299  36
112  10
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 mM e-NAD+, 20 mM EF-2
according to procedure outlined in methods. The data are averages of 2
independent experiments done in triplicates.
117
24530.00
100
A
%
25534.00 26568.00
23713.00
0
20000
21000
22000
23000
24000
25000
26000
27000
28000
29000
24532.00
100
mass
30000
B
%
23700.00
0
20000
21000
22000
23000
24000
25554.00
25000
26000
27000
28000
29000
mass
30000
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.
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.
116
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 b-TAD (PDB entry 1AER) was obtained from the Brookhaven
Protein Data Bank. For more details see Methods chapter.
118
O
NH2
+
N
O
OH
OH
O
O
P O
P O
O
N+
N
O
O
HN
-
-
N
N
O
OH
OH
Figure 39: Structure of e-NAD+.
Excitation: 305 nm; emission: 410 nm