Intrinsic Fluorescence of the Active and the Inactive Functional Forms of Human Thymidylate Synthase

Abstract

The observables associated with protein intrinsic fluorescencespectra, 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.

Intrinsic Fluorometric Reporters of Pteridine Reductase 1, a Target for Antiparasitic Agents

“…Concerning the relationship between protein structure and dynamics and their intrinsic fluorescence properties, extensively quoted reviews are due to Eftink, Engelborghs and Lakowicz [1,6,7]; for very recent specific examples, see Refs. [8][9][10][11]. On the other hand, the exploitation of protein, essentially tryptophan, emission features to monitor and quantitatively characterize protein-ligand binding, including the pitfalls therein, is reviewed and commented in Refs.…”