Nmr Studies Of Biomolecular Structure, Function, And Dyn

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This project utilizes state-of-the-art NMR spectroscopy to study problems that are of continuing interest to the NIEHS, the Laboratory of Structural Biology, and the NMR research group. The primary emphasis currently involves applications in two areas: 1) Understanding how the structural and dynamic behavior of DNA polymerases relates to the fidelity of nucleotide incorporation, and 2) studies of ligand-macromolecule interactions. Recent progress on the first project has included: a) Elucidation of the binding interface for the epsilon and theta subunits of E. coli DNA polymerase III - the main replicative polymerase of E. coli, and b) Determination of the solution structure of the 8 kD lyase domain of human DNA polymerase lambda. The first project involved chemical shift mapping of the amide and methyl resonance shift changes in isotopically labeled epsilon186 that result from the addition of the theta subunit. The most significant shifts observed for the epsilon186 amide resonances are localized to helix alpha1, beta strands 2 and 3, and to the region near the beginning of alpha-helix 7. The determination of the structure of the lyase domain of pol lambda has allowed a structural alignment with the corresponding domain of pol beta. This has allowed us to evaluate previous proposals regarding the involvement of specific residues in the catalytic mechanism. Studies of ligand-macromolecule interactions have focused on: a) understanding the interaction of R67 dihydrofolate reductase - a Type II DHFR, with its substrate and cofactor, and b) characterization of the interaction of borate with trypsin and other enzymes. We were able to obtain interligand NOEs for the ternary R67 DHFR-NADPH-NADP complex that demonstrate the proximity of the NADPH H-4 protons to the NADP H-4 and H-5 protons. We also made the unanticipated discovery that the enzyme is able to catalyze the dephosphorylation of NADP to yield NAD (at a fairly slow rate). This observation provides insight into some of the earlier NMR studies that had been performed on Type II DHFR. We have extended our observations of borate-alcohol-trypsin complexes with additional NMR and crystallographic studies that demonstrate the formation of ternary and quaternary complexes involving borate. This approach provides a potential basis for the development of enzyme inhibitors.