title: Sequence-specific 1H and 15N resonance assignments for human dihydrofolate reductase in solution.
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.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: B J
authors.initials: BJ
authors.lastname: Stockman
authors.forename: N R
authors.initials: NR
authors.lastname: Nirmala
authors.forename: G
authors.initials: G
authors.lastname: Wagner
authors.forename: T J
authors.initials: TJ
authors.lastname: Delcamp
authors.forename: M T
authors.initials: MT
authors.lastname: DeYarman
authors.forename: J H
authors.initials: JH
authors.lastname: Freisheim
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D005492:Folic Acid / Q000378:metabolism; D006801:Humans; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008727:Methotrexate / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D009587:Nitrogen Isotopes; D011487:Protein Conformation; D012996:Solutions; D013329:Structure-Activity Relationship; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics
chemical_list: D009587:Nitrogen Isotopes; D012996:Solutions; D006859:Hydrogen; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate