The long term objective of this work is to understand the structural basis for protein function by studying how changes in structure correlate with changes in function. The structural changes in the protein will be created by changing the amino acid sequence encoded in the structure by various types of mutagenesis, either site specific mutagenesis for producing specific mutants, or site saturation and other approaches for generating families of mutants that can be easily screened for those with a particular function. These approaches will be applied (i) to the enzyme beta-lactamase which is responsible for the frustrating ability of an ever increasing number of strains of infectious microorganisms to resist antibiotic therapy with penicillins and (ii) to the blue copper proteins that are involved in many key redox processes in many organisms. The principle methodologies we shall use include: chemical synthesis of deoxyoligonucleotides, site-directed mutagenesis, various techniques of random mutagenesis, some of which utilize mixtures of oligonucleotides to direct the site(s) of mutagenesis, biophysical observations of the mutant proteins such as rates of catalysis, antibody activation of function, substrate and catalytic specificities, redox and spectroscopic properties, X-ray and nmr determinations of three-dimensional structure. 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 are not hydrolyzed and 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 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 |
