The study of protein-ligand interactions with NMR and fluorescence spectroscopy
Protein-ligand interactions are an extremely important field of study in biochemistry. Information on protein-ligand interactions not only plays an integral role in the fundamental investigation of any enzyme, cellular receptor, or other large biomolecule and the study of its binding properties, but is also important because of its utility in the discovery and development of new drugs. There are many techniques that can be used to study protein-ligand interactions. Widely used techniques include steady-state kinetics (in the presence and absence of potential inhibitors), surface plasmon resonance, mass spectroscopy, a wide array of fluorescent techniques, and various NMR techniques. The studies detailed in this dissertation will focus on the use of one fluorescent technique--a fluorescent polarization competitive displacement assay--and several NMR techniques, including STD (saturation transfer difference) NMR and chemical shift perturbation studies, where either a protein chemical shift is perturbed, or a probe chemical shift when a probe-protein complex is perturbed, upon binding of a potential inhibitor or substrate. Two biochemical systems are focused on in these studies. The bulk of the experiments use NMR spectroscopy to probe the protein-ligand interactions of heme proteins. Cytochromes P450, a family of heme protein enzymes, are an extremely important set of heme proteins, as the human variants are known to be involved in the synthesis of steroid hormones and in the biotransformation of exogeneous compounds--a role essentially like that of a chemical immune system. As such, a characterization of their protein-ligand interactions is deemed especially important in the development of drugs. P450cam, a bacterial P450 that serves as a model system, and P4502B4, one of the membrane-bound, mammalian P450s, serve as the focus of these studies. The other biochemical system studied is that of ERα (alpha-estrogen receptor), a nuclear hormone intracellular receptor. With a growing consensus implicating estrogenic as well as androgenic receptors in the mechanism of endocrine disruption coupled with the fact that ERα serves as the target for a number of breast cancer drugs, it is clear that studies of the protein-ligand interactions of ERα are highly relevant. Together, these studies provide detailed and reliable data concerning the binding properties of several important proteins.
McCullough, Christopher R, "The study of protein-ligand interactions with NMR and fluorescence spectroscopy" (2008). Dissertations (1962 - 2010) Access via Proquest Digital Dissertations. AAI3306519.