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63. Figure 14: Standard curve of e -AMP as measured by fluorescence intensity. - PowerPoint PPT Presentation
Citation preview
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Flu
ores
cenc
e In
tens
ity(A
rbitr
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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.
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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
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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
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1.0
1.2
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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
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-
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1.0
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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
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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
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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.
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Flu
ores
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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
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Flu
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[AF-EF-2] (nM)
94
y y A eo
x x
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b g
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1.0
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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.
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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.
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%
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23713.00 25534.00 26568.00
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%
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