Catalysis and electron transport serve central roles in all biology. The principal investigator aspires to understand the fundamental principles that govern these activities of enzymes (catalysis) and redox proteins (electron transport). The focus is on enzymes (beta-lactamases) that catalyze the hydrolysis of amide bonds (specifically of beta-lactam penicillin type antibiotics), and thereby confer resistance to the therapeutic utility of these antibiotics. Immuno-compromising conditions, such as AIDS and cancer chemotherapy, increase the therapeutic challenge in dealing with an ever increasing number of antibiotic resistant infectious microorganisms. Understanding the way in which the beta- lactamases inactivate beta-lactam antibiotics and the strategies current beta-lactamases might use to adapt in order to inactivate new generations of beta-lactams holds promise of facilitating the development of new more efficacious agents. In photosynthesis in green plants, in the generation of energy during intermediary metabolism, and in a host of other processes, proteins transfer electrons between molecules. The active center in most of these proteins contains a metal; in many of them copper cycles between CuI and CuII. To act effectively in these processes, the metal must possess potentials that are well tuned to the redox processes in which it participates. What features of the proteins create the precise, and sometimes widely varying redox potentials of a metal such as copper? What features of protein structure provide pathways for rapid electron movement? The focus is on two families of copper proteins: the blue copper proteins (such as plastocyanins in green plants where they are involved in photosynthesis or the azurins in bacteria where they are involved in metabolic redox processes) and the copper A center of various oxidases such as a subunit of cytochrome c oxidase which transfers electrons to reduce dioxygen to water as a central step in oxidative phosphorylation. The ultimate aspiration of all of these efforts is to identify which structural features of a protein give rise to various aspects of its function. One can envision at least two important consequences of such insights: (i) the ability to define the activity of a protein of known amino acid sequence which has not been isolated or characterized (the various large scale sequencing projects, including the human genome project) will produce enormous amounts of such information at a rate far exceeding our ability to determine by direct experiment the detailed three-dimensional structure and function of all resulting proteins; and (ii) the ability, using these insights, to design de novo proteins with novel properties. The basic methodologies of these investigations include approaches (for example, site directed, cassette, random, site-saturated mutagenesis) that allow one to create either specific mutants or a wide array of random structural variants of a native protein followed by determination of their functional behavior either using biophysical techniques or by assessing the phenotype the variant protein confers on some biological host.
| Fernandez, C O; Cricco, J A; Slutter, C E et al. (2001) Axial ligand modulation of the electronic structures of binuclear copper sites: analysis of paramagnetic 1H NMR spectra of Met160Gln Cu(A). J Am Chem Soc 123:11678-85 |
