| In this work, we describe the development of computational models to predict nuclear magnetic resonance (NMR) and Mossbauer spectroscopic properties in heme proteins and model systems by using quantum chemical techniques. This work includes density functional theory investigations of the 1H, 13C, 15N and 19F NMR hyperfine shifts, Mossbauer electric field gradient tensors (quadrupole splittings and asymmetry parameters) and their orientations, Mossbauer isomer shifts, and through space J-couplings. Excellent correlations between experiment and theory indicate that the computed wavefunctions are of high quality and molecular orbital analyses provide a graphical representation of the spin density distributions. These powerful new probes are then used in local structure (ligand-binding geometry) refinement and prediction. In part two of this thesis, we describe structural investigations of bisphosphonate drugs and their target enzyme, farnesyl diphosphate synthase (FPPS), using NMR, crystallography and computational techniques. The X-ray structures of nine bisphosphonates are reported together with their 31P solid-state NMR chemical shifts and anisotropic shift (or shielding) tensors, which are highly correlated with the quantum mechanical predictions. Some of these X-ray structures are also used in three-dimensional quantitative structure-activity relationship studies of the bisphosphonate inhibition of Leishmania major FPPS. The FPPS from Trypanosoma brucei (the causative agent of African sleeping sickness) is expressed in E. coli and purified for crystallographic studies. Single crystals of both drug-free and drug-bound FPPSs are obtained and diffract to 3.3 A and 2.5 A, respectively, using synchrotron radiation. A preliminary model of the drug-free enzyme is built using a homology model and the molecular replacement method. Static and electronic structures of bisphosphonates, as well as X-ray structures of FPPS, will further our understanding of FPPS inhibition, allowing for the structure-based design of novel chemotherapeutic agents. |
