| Dihydrofolate Reductases (DHFRs) catalyze the NADPH-dependent reduction of dihydrofolate (DHF), producing tetrahydrofolate (THF), an essential cofactor for many one-carbon transfer cellular reactions. Inhibition of DHFR depletes pools of THF; reducing thymine nucleotides available for DNA synthesis and causing instability and apoptosis. Therefore, DHFRs remain important therapeutic targets. Despite exhaustive investigation of E. coli DHFR, controversy persists over dynamics of regulatory loops and the protonation state of the catalytic ally-important Asp 27 in the apoenzyme and when bound to the inhibitor, methotrexate (MTX). Hydrogen atoms (H) likely play critical roles in DHFR ligand binding and catalysis, yet are difficult to visualize. X-ray and neutron crystallography (NC) have been utilized in this dissertation to provide accurate positions of H within the active site and to probe DHFR dynamics. The ultrahigh resolution X-ray structures of DHFR/MTX, apo DHFR, and DHFR/MTX/NADPH are reported. Novel features are observed in the maps, including electron density for a closed Met20 loop in the apoenzyme (reported disordered previously) and alternate side chain conformations in all structures. The resolution of the apoenzyme and the MTX data allowed anisotropy and full-matrix refinement. From the latter refinement, it was determined that the apoenzyme has highly different bond lengths for its Asp 27 carboxylate, thus, it is neutral at physiological pH whereas the bond lengths of the Asp 27 in the DHFR/MTX crystal are nearly equal, suggesting it is negatively charged.; If H is substituted for deuterium (D), neutrons are especially powerful probes due to their strongly positive scattering. To assign protonation states to the MTX and the Asp 27 by direct identification of D, a 2.2A resolution neutron structure has been solved from data collected on a 0.3mm3 D2O-soaked crystal. DHFR is the 11 th novel protein structure to be solved by NC. Nearly 2/3 of the amide backbone has undergone H/D exchange, an indicator of dynamics. From NC, the MTX N1 is protonated when bound to DHFR, and, paired with X-ray results, this is strong evidence that the Asp 27•MTX interaction is ionic. To decrease background in future neutron experiments, perdeuterated DHFR has been produced, crystallized and analyzed by mass spectrometry. |
