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PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase
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PCR Mutagenesis, Cloning, Expression, Fast Protein Purification Protocols and Crystallization of the Wild Type and Mutant Forms of Tryptophan Synthase

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Human thymidylate synthase with loop 181-197 stabilized in an inactive conformation: ligand interactions,

BeiBei Luo1, Jayanthi Repalli, Al-Motassem Yousef

  • 1Department of Pharmaceutical Sciences, University of South Carolina, Columbia, South Carolina 29208, USA.

Protein Science : a Publication of the Protein Society
|November 11, 2010
PubMed

Insights

Inactive-stabilized thymidylate synthase (TS) mutants exhibit altered structures, substrate responses, and phosphorylation patterns. This suggests a physiological role for TS conformational switching in cancer therapy.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Thymidylate synthase (TS) is a crucial cancer target.
  • TS exhibits conformational switching between active and inactive states.
  • Mutant hTS proteins (M190K, A191K) are stabilized in inactive conformations.

Purpose of the Study:

  • Investigate structural and functional differences of inactive-stabilized hTS mutants.
  • Determine the impact of conformational stabilization on protein phosphorylation.
  • Explore the physiological relevance of TS conformational switching.

Main Methods:

  • Intrinsic fluorescence and circular dichroism spectroscopy.
  • Enzyme kinetics and inhibitor potency assays.
  • Casein kinase 2 (CK2) phosphorylation studies.
  • Temperature dependence analysis of catalysis.

Main Results:

  • Mutants M190K and A191K display unique structural and thermal properties compared to wild-type hTS.
  • Substrate dUMP affects A191K structure but not M190K.
  • M190K shows high CK2 phosphorylation, independent of dUMP.
  • A191K exhibits altered catalytic activity, inhibitor response, and substrate interaction.
  • CK2 phosphorylation of hTS is selective for the inactive conformation.

Conclusions:

  • Inactive-stabilized hTS mutants exhibit distinct biochemical and biophysical characteristics.
  • Protein phosphorylation by CK2 is conformation-specific, highlighting physiological relevance.
  • Conformational switching of TS is a key factor in its biological function and potential therapeutic targeting.

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