Sequence-specific 1H and 15N resonance assignments for human dihydrofolate reductase in solution

Biochemistry. 1992 Jan 14;31(1):218-29. doi: 10.1021/bi00116a031.

Abstract

Dihydrofolate reductase is an intracellular target enzyme for folate antagonists, including the anticancer drug methotrexate. In order to design novel drugs with altered binding properties, a detailed description of protein-drug interactions in solution is desirable to understand the specificity of drug binding. As a first step in this process, heteronuclear three-dimensional NMR spectroscopy has been used to make sequential resonance assignments for more than 90% of the residues in human dihydrofolate reductase complexed with methotrexate. Uniform enrichment of the 21.5-kDa protein with 15N was required to obtain the resonance assignments via heteronuclear 3D NMR spectroscopy since homonuclear 2D spectra did not provide sufficient 1H resonance dispersion. Medium- and long-range NOE's have been used to characterize the secondary structure of the binary ligand-enzyme complex in solution.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Folic Acid / metabolism
  • Humans
  • Hydrogen
  • Magnetic Resonance Spectroscopy / methods
  • Methotrexate / chemistry
  • Molecular Sequence Data
  • Mutation
  • Nitrogen Isotopes
  • Protein Conformation
  • Solutions
  • Structure-Activity Relationship
  • Tetrahydrofolate Dehydrogenase / chemistry*
  • Tetrahydrofolate Dehydrogenase / genetics

Substances

  • Nitrogen Isotopes
  • Solutions
  • Hydrogen
  • Folic Acid
  • Tetrahydrofolate Dehydrogenase
  • Methotrexate