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Dimer–monomer equilibrium of human thymidylate synthase monitored by fluorescence resonance energy transfer
Author(s) -
Genovese Filippo,
Ferrari Stefania,
Guaitoli Giambattista,
Caselli Monica,
Costi M. Paola,
Ponterini Glauco
Publication year - 2010
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.379
Subject(s) - förster resonance energy transfer , thymidylate synthase , chemistry , allosteric regulation , dimer , fluorescence , bioconjugation , monomer , atp synthase , enzyme , stereochemistry , biochemistry , biology , fluorouracil , physics , organic chemistry , chemotherapy , quantum mechanics , genetics , polymer
Abstract An ad hoc bioconjugation/fluorescence resonance energy transfer (FRET) assay has been designed to spectroscopically monitor the quaternary state of human thymidylate synthase dimeric protein. The approach enables the chemoselective engineering of allosteric residues while preserving the native protein functions through reversible masking of residues within the catalytic site, and is therefore suitable for activity/oligomerization dual assay screenings. It is applied to tag the two subunits of human thymidylate synthase at cysteines 43 and 43′ with an excitation energy donor/acceptor pair. The dimer–monomer equilibrium of the enzyme is then characterized through steady‐state fluorescence determination of the intersubunit resonance energy transfer efficiency.