STRUCTURE-FUNCTION STUDIES OF DIHYDROFOLATE REDUCTASE

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This project is concerned with various aspects of the relation of the structure to the function of dihydrofolate reductase (DHFR). The proposed problems to be investigated include: (a) site- directed mutagenesis studies of human DHFR Using recombinant DNA techniques to evaluate the functional role of various NADPH and methotrexate (MTX) or dihydrofolate (FAH2) binding amino acid residues. These studies will involve construction of recombinant plasmids and high level DHFR expression in E. coli, purification and characterization of mutant forms of human DHFR by kinetic, equilibrium binding and x-ray crystallographic studies; (b) mapping of the immunogenic determinants of human DHFR using antibodies to peptides produced by proteolysis or chemical cleavage of intact human DHFR or produced by automated peptide synthesis. These sequence-specific antibodies will also be used to detect MTX-insensitive DHFRs from human acute myelogenous leukemic cells and other tumors; (c) to obtain sequence information on a MTX-insensitive DHFR from L5178Y cells; (d) evaluation of newly synthesized folate antagonists as inhibitors of DHFRs and thymidylate synthase and as cytotoxic agents; (e) molecular characterization of the one-carbon FAH4/MTX transport system in transport-competent and - defective L1210 cells. These experiments include photoaffinity labeling of transport proteins with an MTX analogue under various conditions, a determination of the relatedness of the membrane and cytoplasmic MTX binding components, possible expression of components in transport-defective cells and sequencing of a transport protein in L1210 cells which overproduce this protein.