Biophysical studies of Thiopurine
S-methyltransferase (TPMT)
variants
Paolo Dametto September 2009
TPMT
• TPMT (thiopurine S-methyltransferase) is a cytosolic
enzyme that catalyzes the S-methylation of aromatic
and heterocyclic sulfhydril compounds, including
drugs such as thiopurines.
Thiopurines 6-mercaptopurine (6-MP), 6-thioguanine (6-TG), and azathiopurine (AZA) are thiopurine drugs.
• lymphoblastic leukemia (ALL)
• inflammatory bowel disease (IBD) • autoimmune disorders • organ transplant recipient
6-MP 6-TG AZA
TPMT structure
TPMT Variant
Amino acid change Structural context
Enzyme Activity
*1 WT
*2 A80P Helix Low
*3A A154T/Y240C b-Strand Low
*3B A154T b-Strand Low
*3C Y240C b-Strand Low
*3D Stopp/A154T/Y240C Low
*4 Splicing Low
*5 L49S Helix Low
*6 Y180F b-Strand Low
*7 H227Q Helix Low
*8 R215H b-Strand Intermediate
*9 K119T b-Strand Intermediate
*10 G144R Low
*11 C132Y b-Strand Low
*12 S125L Turn Low
*13 E28V Helix Low
*14 Deletion Low
*15 Deletion Low
*16 R163H Helix Intermediate
*17 Q42E Intermediate
*18 G71R Turn Intermediate
*19 K122T b-Strand High
*20 K238E b-Strand
*21 L69V Turn
*22 R163P Helix
*23 A167G Helix High/low??
*24 Q179H b-Strand
*25 C212R Turn
TPMT variants
Position of TPMT*2, *3C, *5 mutations
*3C (Tyr240→Cys) *2 (Ala80→Pro)
*5 (Leu49→Ser)
Purification’s step……
1. TPMT large scale expression in BL21/DE3 cells
2. Sonication
3. His-Tag purification using Ni-NTA column
4. Removing His-Tag sequence with Biotinylated Thrombin
5. His-Tag purification
6. Gel Filtration chromatography
CD: circular dichroism
The FAR-UV (190 ÷ 260 nm) CD spectrum can reveal important characteristics of proteins.
• estimation of secondary structure
• molecule changes in the secondary structure as a function of temperature or of the concentration of denaturing agents
• thermodynamic information such as ∆G and Tm
-30000
-25000
-20000
-15000
-10000
-5000
0
5000
10000
15000
20000
25000
30000
35000
190 200 210 220 230 240 250 260 270
Resid
ue e
llipt
icity
[deg
rees
*cm
2*dm
ol-1
]
Wavelength [nm]
TPMT*1
TPMT*2
TPMT*3C
TPMT*5
Secondary structure analysis
Thermal unfolding analysis (222 nm)
-13000
-12000
-11000
-10000
-9000
-8000
-7000
-6000
-5000
-4000
-3000
-2000
-1000
010 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85
Resid
ue e
llipt
icity
[deg
rees
*cm
2*dm
ol-1
]
Temperature [°C] TPMT*1 TPMT*2 TPMT*3C TPMT*5
From Thermal Denaturation Analysis FAR UV_222nm
Protein Tm [°C] ∆G(25°C) [Kcal/mol]
∆(∆G) [Kcal/mol]
∆Sm [J/mol*K]
∆Hm [Kcal/mol]
TPMT*1 58.9 14.5 // -1788.4 141.9
TPMT*2 32.8 1.27 11.19 -549.9 40.2
TPMT*3C 42.9 3.77 6.84 -879.8 66.4
TPMT*5 49.3 7.66 4.14 -1318.7 101.6
Thermodynamic parameters
ANS •The aromatic chromophore 1-anilino-8-naphthalene sulfonate (ANS) is feebly fluorescent in water but the intensity is dramatically increased in nonpolar solvent or when it binds to nonpolar sites of proteins.
•ANS was used to check if the TPMT variants showed some hydrophobic pattern, according to their enhanced thermodynamic instability. In this case, the ANS signal would be stronger than that of TPMT*1.
-500
500
1500
2500
3500
4500
5500
6500
7500
8500
9500
10500
11500
12500
0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 2.5 2.75
Fluo
resc
ence
Inte
nsity
GdnCl [M]
TPMT*1
TPMT*2
TPMT*3C
TPMT*5
ANS fluorescence measurements at 478 nm with a gradient of GdnCl
!
-500000
500000
1500000
2500000
3500000
4500000
5500000
6500000
7500000
8500000
9500000
10500000
400 420 440 460 480 500 520 540 560 580 600
Fluo
resc
ence
inte
sity
Wavelength [nm]
0 µM SAM
100 µM SAM
200 µM SAM
1 mM SAM
3 mM SAM
ANS fluorescence measurements of TPMT*1 at different concentrations of SAM
SAM/ANS binding site
ANS binding site
SAM/ANS binding site
ANS binding site
SAM/ANS binding site
-500000
500000
1500000
2500000
3500000
4500000
5500000
6500000
7500000
8500000
9500000
10500000
11500000
400 420 440 460 480 500 520 540 560 580 600
Fluo
resc
ence
inte
nsity
Wavelength [nm]
TPMT*1 no SAM
TPMT*2 no SAM
TPMT*3C no SAM
TPMT*5 no SAM
ANS fluorescent measurements
Protein Enzyme activity at 37 °C Enzyme activity at 18 °C
TPMT *1 (WT) 100% 100%
TPMT *2 (A80P) 35% 48%
TPMT *5 (L49S) 0% 14%
Enzyme activity
Limited proteolysis
Limited proteolysis can be used to probe conformational features of protein. • Limited proteolysis and matrix-assisted laser
desorption/ionization mass spectrometry (MALDI-TOF) was applied to probe protease-accessible sites of TPMT.
• Fragments were analyzed using the software MTMDAT.
0
0
0.05
0.1
0.15
0.2
0.25
0.3
17 24 31 38 45 52 59 66 73 80 87 94
101
108
115
122
129
136
143
150
157
164
171
178
185
192
199
206
213
220
227
234
TPMT*1
0
0
0.05
0.1
0.15
0.2
0.25
0.3
17 24 31 38 45 52 59 66 73 80 87 94
101
108
115
122
129
136
143
150
157
164
171
179
186
193
200
207
214
221
228
235
TPMT*2
0
0
0.05
0.1
0.15
0.2
0.25
0.3
17 23 29 35 41 47 53 59 65 71 77 83 89 9510
110
711
311
912
513
113
714
314
915
516
116
717
317
918
519
119
720
320
921
522
122
723
323
9
TPMT*1
0
0
0.05
0.1
0.15
0.2
0.25
0.3
17 23 29 35 41 47 53 59 65 71 77 83 89 9510
110
711
311
912
513
113
714
314
915
516
116
717
317
918
519
119
720
320
921
522
122
723
323
9
TPMT*5
TPMT*2 TPMT*5
Considerations • The TPMT protein can broaden our
understanding of the mechanisms by which common polymorphisms can lead to functional effects.
• TPMT protein represents one of the most
striking example of the science named Pharmacogenomics.
Thank you for listening
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