Biophysical studies of Thiopurine S-methyltransferase (TPMT) variants
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This document describes biophysical studies of Thiopurine S-methyltransferase (TPMT) variants. TPMT is an enzyme that metabolizes thiopurine drugs used to treat conditions like leukemia. The document identifies several naturally occurring TPMT variants with reduced or intermediate enzyme activity. It then details experiments analyzing the structure, stability, and activity of wild type and variant TPMT proteins using techniques like circular dichroism, thermal unfolding, limited proteolysis, and enzyme activity assays. The results provide insights into how common TPMT variants alter the protein's structure and function, affecting thiopurine drug metabolism.
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Biophysical studies of Thiopurine S-methyltransferase (TPMT) variants
- 1. Biophysical studies of Thiopurine S-methyltransferase (TPMT) variants Paolo Dametto September 2009
- 2. 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.
- 3. 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
- 5. TPMT variants TPMT Variant Amino acid change Structural context Enzyme Activity *1 WT *2 A80P Helix Low *3A A154T/Y240C b-Strand Low *14 Deletion Low *3B A154T b-Strand Low *15 Deletion Low *3C Y240C b-Strand Low *16 R163H *3D Stopp/A154T/Y240C Low *4 Splicing Low *17 Q42E *5 L49S Helix Low *18 G71R Turn Intermediate *6 Y180F b-Strand Low *19 K122T b-Strand High *7 H227Q Helix Low *20 K238E b-Strand *8 R215H b-Strand Intermediate *21 L69V Turn *22 R163P Helix *23 A167G Helix b-Strand Helix Intermediate Intermediate *9 K119T Intermediate *10 G144R *11 C132Y b-Strand Low *12 S125L Turn Low *24 Q179H b-Strand *13 E28V Helix Low *25 C212R Turn Low High/low??
- 6. Position of TPMT*2, *3C, *5 mutations *2 (Ala80→Pro) *3C (Tyr240→Cys) *5 (Leu49→Ser)
- 7. 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
- 8. 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
- 9. Secondary structure analysis 35000 Residue ellipticity [degrees*cm2*dmol-1] 30000 TPMT*1 25000 TPMT*2 20000 TPMT*3C 15000 TPMT*5 10000 5000 0 -5000 190 200 210 220 230 -10000 -15000 -20000 -25000 -30000 Wavelength [nm] 240 250 260 270
- 10. Thermal unfolding analysis (222 nm) 10 15 20 25 30 35 40 45 50 55 60 65 Residue ellipticity [degrees*cm2*dmol-1] 0 -1000 -2000 -3000 -4000 -5000 -6000 -7000 -8000 -9000 -10000 -11000 -12000 -13000 Temperature [°C] TPMT*1 TPMT*2 TPMT*3C TPMT*5 70 75 80 85
- 11. Thermodynamic parameters 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
- 12. 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.
- 13. ANS fluorescence measurements at 478 nm with a gradient of GdnCl 12500 11500 ! 10500 Fluorescence Intensity 9500 8500 TPMT*1 TPMT*2 TPMT*3C 7500 TPMT*5 6500 5500 4500 3500 2500 1500 500 -500 0 0.25 0.5 0.75 1 1.25 1.5 GdnCl [M] 1.75 2 2.25 2.5 2.75
- 18. ANS fluorescence measurements of TPMT*1 at different concentrations of SAM 10500000 0 µM SAM 100 µM SAM 8500000 200 µM SAM 7500000 Fluorescence intesity 9500000 1 mM SAM 6500000 3 mM SAM 5500000 4500000 3500000 2500000 1500000 500000 -500000 400 420 440 460 480 500 Wavelength [nm] 520 540 560 580 600
- 20. ANS binding site SAM/ANS binding site
- 21. ANS binding site SAM/ANS binding site
- 22. ANS fluorescent measurements 11500000 10500000 TPMT*1 no SAM 8500000 Fluorescence intensity 9500000 TPMT*2 no SAM TPMT*3C no SAM 7500000 TPMT*5 no SAM 6500000 5500000 4500000 3500000 2500000 1500000 500000 -500000 400 420 440 460 480 500 520 Wavelength [nm] 540 560 580 600
- 23. Enzyme activity 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%
- 24. 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.
- 27. TPMT*2 TPMT*5
- 28. 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.
- 29. Thank you for listening