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

• Published in: Biosensors & bioelectronics Vol. 64; pp. 36 - 42
• Main Authors: Antosiewicz, Anna, Senkara, Elżbieta, Cieśla, Joanna
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 15-02-2015
• Subjects: Binding energy; Biosensing Techniques - methods; Biosensors; Complex formation; Dihydrofolate reductase; Dissipation; Enzymes; Humans; Kinetics; Microbalances; Microscale thermophoresis; Multienzyme Complexes - chemistry; Multienzyme Complexes - metabolism; Protein complex; Protein Interaction Mapping - methods; Quartz crystal microbalance; Quartz Crystal Microbalance Techniques - methods; Synthesis; Tetrahydrofolate Dehydrogenase - chemistry; Tetrahydrofolate Dehydrogenase - metabolism; Thermodynamics; Thermophoresis; Thymidylate biosynthesis; Thymidylate synthase; Thymidylate Synthase - chemistry; Thymidylate Synthase - metabolism; Protein complex; Thymidylate biosynthesis; Quartz crystal microbalance; Dihydrofolate reductase; Thymidylate synthase; Microscale thermophoresis
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2014.08.031

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 10µM (both methods), kon=0.46×104M−1s−1 and koff=0.024s−1 (QCM-D). We also calculated Gibbs free energy as in the order of −30kJ/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.
•Thymidylate synthesis cycle enzymes are important chemotherapy targets.•Human TS and DHFR interaction has been studied by the first time by means of QCM-D and MST.•QCM-D and MST provided kinetic parameters and stoichiometry of the studied TS-DHFR complex.•MST enabled the characterization of the studied complex in the aspect of binding cooperativity.•QCM-D and MST gave comparable and complementary results in protein–protein interactions studies.