The long term objective of this work is to understand the molecular basis for protein function by gaining insights into how changes in structure correlate with changes in function. We will create structural variations in the protein by altering the amino acid sequence encoded by the DNA. For this purpose we shall employ various techniques that allow specific changes to be introduced at a single site (for example, site directed mutagenesis) or many sites concurrently (for example, cassette mutagenesis). We will also generate families of mutants (for example, by cassette mutagenesis that incorporates many codons at particular sites of interest); these families can then be screened for those whose functions have changed in interesting ways. We will apply these approaches to two families of proteins: (i) the enzymes, Beta-lactamases, that are responsible for the frustrating ability of an ever increasing number of strains of infectious microorganisms to resist antibiotic therapy with penicillins; (ii) the blue copper proteins that are involved in key redox processes in many organisms, including photosynthesis in green plants. The principle methodologies we shall use include: many techniques of mutagenesis, screening for the phenotype with which various mutants endow their bacterial hosts, biophysical observations of the mutant proteins after isolation and purification such as determination of the rates of catalysis on substrates of differing structure, redox and spectroscopic properties, three-dimensional structure by X-ray diffraction. To understand the mechanisms of action of Beta-lactamase should be of great importance in allowing the design of new antibiotics of the penicillin/cephalosporin type that cannot be hydrolyzed and thereby inactivated by the enzyme. This should provide powerful new agents to combat infections by the many strains of microorganisms that are presently resistant to many penicillin-type antibiotics. To understand the origin of the well-tuned redox properties of blue copper proteins will reveal new insights into how the ligand environment determines the electronic properties of the bound metal, how electron transfer occurs in many key biological processes such as photosynthesis; the blue copper mutants will further provide valuable models in which to test fundamental aspects of electron transfer between two metals. To be able to create and study many structural analogues of a parent protein will provide striking new insights into how proteins carry out their myriad functions (as, for example, biological catalysts, hormones, transport agents, cell surface receptors, structural elements in cells and organisms, muscles that convert chemical energy into work, antibodies that distinguish self from non-self); it will also give us eventually the ability to design proteins with specific, novel and useful properties.
| 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 |
