NMR spectroscopy and its biological applications; structure function relationships in proteins; stable-isotope-assisted multinuclear magnetic resonance spectroscopy; processing and analysis of multi-dimensional NMR data; structural genomics; metabolomics
The central theme of our research is the application of nuclear magnetic resonance (NMR) spectroscopy to the solution of biochemical problems. The unique power of NMR lies in its ability to provide detailed chemical and structural information at an atomic level about molecules in solution--even when they are present in living cells or organisms. The general strategy is to use multidimensional (2D, 3D, and 4D), multinuclear magnetic resonance techniques to detect and assign resonances from atoms of biological interest (e.g., 1H, 13C, 15N, and 31P). With these assignments in hand, we can then interpret the wealth of spectral information present in coupling constants, relaxation rates, cross-relaxation rates, and chemical shifts. Proton-proton cross-relaxation rates and a variety of measured coupling constants are used to derive three-dimensional structures of these macromolecules. Relaxation rates, line-shapes, and nuclear Overhauser effect measurements provide information about molecular motions and conformational changes. The kinds of information gained from such investigations can be critical for learning how these molecules work and how they can be redesigned to have desired properties. Our work focuses on protein systems: Enzymes, electron transport proteins, proteinase inhibitors, and nucleic acid binding proteins. We exploit recombinant DNA technology as a means for producing the large amounts of protein needed for NMR investigations and for introducing stable isotopes of interest (most commonly 2H, 13C, and 15N). Mutagenesis studies allow us to test hypotheses about the roles of individual amino acid residues in determining properties such as local structure, conformations and mobilities of side chains, hydrogen exchange kinetics, rates of protein folding or unfolding, pKavalues, oxidation-reduction potentials, and ligand binding.
Graduate students and postdoctoral fellows in the laboratory usually focus on a particular biochemical system and use NMR as one of the tools for its analysis. They are expected to become experienced in preparing samples and in carrying out functional studies. Alternatively, they may focus on developing instrumentation or novel ways of collecting or analyzing NMR data.
Backbone ribbon diagram of brazzein indicating the positions of residues found to be critical for the sweetness of the protein. Color code: red, enhanced sweetness; light blue, moderately decreased sweetness; dark blue, strongly decreased sweetness; dark gray, sweetness equivalent to wild-type; light gray, residues not yet mutated (Assadi-Porter et al., 2000).