title: Quartz crystal microbalance with dissipation and microscale thermophoresis as tools for investigation of protein complex formation between thymidylate synthesis cycle enzymes.
abstract: 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), k(on) = 0.46 × 10(4) M(-1) s(-1) and k(off) = 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.
authors.affiliation: Institute of Biotechnology, Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland. Electronic address: aantosiewicz@ch.pw.edu.pl.
authors.forename: Anna
authors.initials: A
authors.lastname: Antosiewicz
authors.affiliation: Institute of Biotechnology, Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland. Electronic address: esenkara@ch.pw.edu.pl.
authors.forename: Elżbieta
authors.initials: E
authors.lastname: Senkara
authors.affiliation: Institute of Biotechnology, Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw, Poland. Electronic address: jciesla@ch.pw.edu.pl.
authors.forename: Joanna
authors.initials: J
authors.lastname: Cieśla
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D006801:Humans; D007700:Kinetics; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*; D025941:Protein Interaction Mapping / Q000379:methods; D058525:Quartz Crystal Microbalance Techniques / Q000379:methods*; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D013816:Thermodynamics; D013940:Thymidylate Synthase / Q000737:chemistry / Q000378:metabolism*
chemical_list: D009097:Multienzyme Complexes; C056930:thymidylate synthase-dihydrofolate reductase; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase
keywords: Dihydrofolate reductase; Microscale thermophoresis; Protein complex; Quartz crystal microbalance; Thymidylate biosynthesis; Thymidylate synthase