We propose to apply multinuclear solid state NMR techniques to study the structure and dynamics of E. coli R67 plasmid, E. coli chromosomal and chicken liver dihydrofolate reductases (DHFR). DHFR's are small, biochemically and pharmaceutically important enzymes whose structures have been solved to high resolutions (1.7 angstrom). Both the E. coli chromosomal and plasmid enzymes can be prepared in large quantities from genetically manipulated DHFR overproducing strains. The long term objectives of this project are to obtain detailed structure and dynamics information of several bacteria and vertibrate DHFR's and their ligands, and to deduce the relationship between structure, dynamics and functions of DHFR's. Initially we will focus on the antibacteria drug, trimethoprim (TMP), and the smallest DHFR, the type II enzyme from E. coli R67 plasmid (M.W. approximately 8500). TMP will be studied in pure form and in binary and ternary complexes with the three DHFR's mentioned above, and with NADPH. The structure and dynamics information will be deduced from analyses of various measured dipolar, quadrupolar and the anisotropic chemical shift interactions, and relaxation rates. To facilitate data analysis, both TMP and DHFR will be labelled with various isotopes at specific sites. NMR samples will be in either single crystal or polycrystalline forms. (R plasmid DHFR single crystal of 1.2 x 0.6 x 0.6mm was grown in 24 hours.). Theoretically calculated NMR spectra of DHFR single crystals, based on coordinates determined by x-ray crystallographic method, will be compared and contrasted with NMR results. Discrepancies between them, if detected, will be analyzed to deduce the structure and dynamics of DHFR. Final data analysis will include modeling of DHFR-ligand interactions on an Evans and Sutherland molecular graphic system. Completion of this project is expected to provide better understanding on the roles of molecular dynamics and the function of DHFR's. Our data will also fill the gap of providing electronic and dynamical information which are needed for theoretical and empirical approaches in designing effective drugs.
| Yang, Q X; Huang, F Y; Huang, T H et al. (1993) The effect of hydration on the dynamics of trimethoprim bound to dihydrofolate reductase. A deuterium NMR study. Biophys J 64:1361-5 |
| Huang, T H; DeSiervo, A J; Yang, Q X (1991) Effect of cholesterol and lanosterol on the structure and dynamics of the cell membrane of Mycoplasma capricolum. Deuterium nuclear magnetic resonance study. Biophys J 59:691-702 |
| Huang, F Y; Yang, Q X; Huang, T H et al. (1991) The conformations of trimethoprim/E. coli dihydrofolate reductase complexes. A 15N and 31P NMR study. FEBS Lett 283:44-6 |
| Huang, F Y; Yang, Q X; Huang, T H (1991) 15N NMR studies of the conformation of E. coli dihydrofolate reductase in complex with folate or methotrexate. FEBS Lett 289:231-4 |
