Quartz crystal microbalance with dissipation and microscale thermophoresis as tools for investigation of protein complex formation between thymidylate synthesis cycle enzymes
Anna Antosiewicz , Elżbieta Senkara-Barwijuk , Joanna Cieśla
AbstractAbstract Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) play essential role in DNA synthesis, repair and cell division by catalyzing two subsequent reactions in thymidylate biosynthesis cycle. The lack of either enzyme leads to thymineless death of the cell, therefore inhibition of the enzyme activity is a common and successful tool in cancer chemotherapy and treatment of other diseases. However, the detailed mechanism of thymidylate synthesis cycle, especially the interactions between cycle enzymes and its role remain unknown. In this paper we are the first to show that human TS and DHFR enzymes form a strong complex which might be essential for DNA synthesis. Using two unique biosensor techniques, both highly sensitive to biomolecular interactions, namely quartz crystal microbalance with dissipation monitoring (QCM-D) and microscale thermophoresis (MST) we have been able to determine DHFR–TS binding kinetic parameters such as the Kd value being below 10 µM (both methods), kon=0.46×104 M−1 s−1 and koff=0.024 s−1 (QCM-D). We also calculated Gibbs free energy as in the order of −30 kJ/mol and DHFR/TS molar ratio pointing to binding of 6 DHFR monomers per 1 TS dimer (both methods). Moreover, our data from MST analysis have pointed to positive binding cooperativity in TS–DHFR complex formation. The results obtained with both methods are comparable and complementary.
|Journal series||Biosensors & Bioelectronics, ISSN 0956-5663, (A 40 pkt)|
|Publication size in sheets||0.5|
|Keywords in English||Quartz crystal microbalance; Microscale thermophoresis; Protein complex; Thymidylate synthase; Dihydrofolate reductase; Thymidylate biosynthesis|
|Score|| = 40.0, 28-11-2017, ArticleFromJournal|
= 40.0, 28-11-2017, ArticleFromJournal
|Publication indicators||: 2015 = 7.476 (2) - 2015=6.675 (5)|
|Citation count*||10 (2018-11-13)|
* presented citation count is obtained through Internet information analysis and it is close to the number calculated by the Publish or Perish system.