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 inter-subunit resonance energy transfer efficiency.

Dimer-monomer equilibrium of human thymidylate synthase monitored by fluorescenceresonance energy transfer / Genovese, Filippo; Ferrari, Stefania; Guaitoli, Giambattista; Caselli, Monica; Costi, Maria Paola; Ponterini, Glauco. - In: PROTEIN SCIENCE. - ISSN 0961-8368. - STAMPA. - 19:5(2010), pp. 1023-1030. [10.1002/pro.379]

Dimer-monomer equilibrium of human thymidylate synthase monitored by fluorescenceresonance energy transfer.

GENOVESE, Filippo;FERRARI, Stefania;GUAITOLI, GIAMBATTISTA;CASELLI, Monica;COSTI, Maria Paola;PONTERINI, Glauco
2010

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 inter-subunit resonance energy transfer efficiency.
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1030
Dimer-monomer equilibrium of human thymidylate synthase monitored by fluorescenceresonance energy transfer / Genovese, Filippo; Ferrari, Stefania; Guaitoli, Giambattista; Caselli, Monica; Costi, Maria Paola; Ponterini, Glauco. - In: PROTEIN SCIENCE. - ISSN 0961-8368. - STAMPA. - 19:5(2010), pp. 1023-1030. [10.1002/pro.379]
Genovese, Filippo; Ferrari, Stefania; Guaitoli, Giambattista; Caselli, Monica; Costi, Maria Paola; Ponterini, Glauco
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11380/639982
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