|
|
|
|
Dr. ReinhartThe Reinhart laboratory is interested in understanding the molecular basis for enzyme regulation, particularly regulation that results from the binding of allosteric ligands. We are currently studying phosphofructokinase from several different bacterial sources as well as carbamoyl phosphate synthetase from E. coli. Each of these enzymes has an important role to play in the cell, not only in establishing the flux through their respective metabolic pathways, but also in allowing that flux to change in response to changing environmental and metabolic circumstances. The question we address is what structural features within the proteins allow these enzymes to be responsive in the way that specifically suits their physiological role. We are interested in dynamic and well as static elements of structure, and consequently much of our efforts are focussed on obtaining data that are complementary to structural information derived from X-ray crystallography in order to develop a more comprehensive picture of the molecular basis of this regulatory functionality. A wide variety of experimental techniques and procedures are used in the Reinhart laboratory, including bacterial cell culturing, protein purification, site-directed mutagenesis, high performance chromatography, steady-state enzyme kinetics, pre-steady-state protein-ligand binding kinetics using stopped-flow, isothermal titration calorimetry, steady- state and time resolved fluorescence spectroscopy and fluorescence anisotropy, high-pressure manipulation of macromolecular equilibria, two-photon fluctuation correlation spectroscopy, and molecular modeling. Consequently many different types of projects can be developed depending on the experience the student has had in the past and desires to experience in the future. Examples of specific projects include (but are not limited to): 1) assessing the contribution made by each of the six tryptophans to the overall intrinsic fluorescence of carbamoyl phosphate synthetase by examining mutants in which tryptophans have been changed individually to tyrosine via site-directed mutagenesis; 2) construction of a mutant form of phosphofructokinase that would form an intersubunit disulfide bond; 3) production and characterization of a phosphofructokinase containing a tryptophan analog with altered fluorescence properties in place of the single native tryptophan; 4) systematically assessing the relative stability of mutants of phosphofructokinase to establish regions of the proteins most resistant to destabilization upon site-directed mutagenesis. |
