Quartz crystal microbalance with dissipation and microscale thermophoresis as tools for investigation of protein complex formation between thymidylate synthesis cycle enzymes

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.