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Biophysical characterization of the *5 protein variant of human thiopurine methyltransferase by NMR spectroscopy
Linköping University, Department of Physics, Chemistry and Biology, Molecular Biotechnology.
2012 (English)Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
Abstract [en]

Human thiopurine methyltransferase (TPMT) is an enzyme involved in the metabolism of thiopurine drugs, which are widely used in leukemia and inflammatory bowel diseases such as ulcerative colitis and Crohn´s disease. Due to genetic polymorphisms, approximately 30 protein variants are present in the population, some of which have significantly lowered activity. TPMT *5 (Leu49Ser) is one of the protein variants with almost no activity. The mutation is positioned in the hydrophobic core of the protein, close to the active site.

Hydrogen exchange rates measured with NMR spectroscopy for N-terminally truncated constructs of TPMT *5 and TPMT *1 (wild type) show that local stability and hydrogen bonding patterns are changed by the mutation Leu49Ser. Most residues exhibit faster exchange rates and a lower local stability in TPMT *5 in comparison with TPMT *1. Changes occur close to the active site but also throughout the entire protein. Calculated overall stability is similar for the two constructs, so the measured changes are due to local stability.

Protein dynamics measured with NMR relaxation experiments show that both TPMT *5 and TPMT *1 are monomeric in solution. Millisecond dynamics exist in TPMT *1 but not in TPMT *5, even though a few residues exhibit a faster dynamic. Dynamics on nanosecond to picosecond time scale have changed but no clear trends are observable.

Place, publisher, year, edition, pages
2012. , 43 p.
Keyword [en]
thiopurine methyltransferase, hydrogen exchange, nuclear magnetic resonance, relaxation, protein dynamics, local stability
National Category
Structural Biology
URN: urn:nbn:se:liu:diva-78526ISRN: LITH-IFM-A-EX--12/2583--SEOAI: diva2:533774
Subject / course
Chemical Biology
Physics, Chemistry, Mathematics
Available from: 2012-06-18 Created: 2012-06-14 Last updated: 2012-06-18Bibliographically approved

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