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Intrinsic Fluorescence of the Active and the Inactive Functional Forms of Human Thymidylate Synthase
Author(s) -
Vitiello Simone,
Caselli Monica,
Pavesi Giorgia,
Santucci Matteo,
Ferrari Stefania,
Paola Costi Maria,
Ponterini Glauco
Publication year - 2021
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.202000722
Subject(s) - thymidylate synthase , chemistry , biophysics , fluorescence , enzyme , monomer , kinetics , fluorescence anisotropy , enzyme kinetics , biochemistry , active site , combinatorial chemistry , stereochemistry , biology , cancer , fluorouracil , genetics , physics , organic chemistry , quantum mechanics , membrane , polymer
The observables associated with protein intrinsic fluorescence – spectra, time decays, anisotropies – offer opportunities to monitor in real time and non‐invasively a protein‘s functional form and its interchange with other forms with different functions. We employed these observables to sketch the fluorometric profiles of two functional forms of human thymidylate synthase (hTS), a homodimeric enzyme crucial for cell proliferation and thus targeted by anticancer drugs. The protein takes an active and an inactive form. Stabilization of the latter by peptides that, unlike classical hTS inhibitors, bind it at the monomer/monomer interface offers an alternative inhibition mechanism that promises to avoid the onset of drug resistance in anticancer therapy. The fluorescence features depicted herein can be used as tools to identify and quantify each of the two protein forms in solution, thus making it possible to investigate the kinetic and thermodynamic aspects of the active/inactive conformational interchange. Two examples of fluorometrically monitored interconversion kinetics are provided.