Research Topics
Department of Biochemistry
Ph.D. (1975) University of Illinois
Research Topics
Research Interests Mechanisms of biological oxidation, the structure
of collective assemblies of organized protein and nucleic acid systems, the
basis of molecular recognition in protein-protein and protein-nucleic acid complexes,
and the detailed chemistry and physical operation of oxygenase and odxidase
catalysis.
One research focus is on the cytochrome P-450 dependent mixed function oxidases which play central and crucial roles in mammalian, plant, insect, viral, and microbial metabolism. Biotransformations catalyzed by the P-450 monoxygenases of primary medical relevance include the detoxification of ingested toxins and pollutants, carcinogen activation and deactivation as carried out in the human liver and lung, and steroid hormone biosynthesis in the adrenals, testes, and ovaries. All P-450 catalyzed monoxoygenase reactions reduce atmospheric dioxygen producing water and a monoxygenated substrate. Central questions relating to the mechanisms of these important biological oxidations include the precise chemistry involved in activation of oxygen and substrate and the identity of metal-oxygen-carbon intermediates in the catalytic event, the detailed physical description of inter- and intra-protein electron and proton transfer, and the structure of multi-enzyme membrane complexes involved in catalytic oxygenation and redox movement.
A second important research direction for our group is the development and execution of methodologies for the determination of biological structures in the 5 nm -500 ‘mesocale’ size range, and determination of information defining the collective properties of organized assemblies of biological macromolecules and their role in determining function. Perhaps the most interesting processes in biology utilize complexes of protein, nucleic acid, lipid, and sugar that occupy this mesoscale domain. Providing the important structural and functional information of specific aggregates of nucleic acid and protein which make up ribosomes, spliceosomes, and transcription complexes is an important goal of our research. Toward this end we use the latest advances in atomic probe imaging, including scanning tunneling (STM), atomic force (AFM) and near field optical (NSOM) microscopies to investigate central and significant problems in cell and molecular biology. Recently we have been able to directly image single high density lipoproteins (HDL) and the AFM. These particles, the so-called “good-cholesterol” are involved in the transport of cholesterol esters from the peripheral vasculature to the liver. Single HDL particles were found to be discoidal in shape, with a height corresponding to a single lipid bilayer of 5.5 nm and a width of between 10 nm and 20 nm depending on the stoichiometry of protein to lipid. Single copies of the enzyme lecithin cholesterol acyl transferase bound to an individual disk have been observed under physiological conditions. The interactions of HDL particles with surfaces, receptors and enzymes of cholesterol metabolism are providing important new information on the mechanisms of atherosclerosis. The HDL particles are also proving to be beautiful nanoscale supported lipid bilayers for the selective incorporation and study of single ion conducting channels and membrane associated enzymes. A second system under investigation with the AFM is the spliceosome. This well-defined protein - nucleic acid complex controls the recognition and excision of intron mRNA segments. Most exciting is the potential of identifying the unique topologies indicated in the accompanying schematic with atomic probe imaging methods thereby defining for the first time the functional aspects of the mesoscale assemblies involved in RNA processing.
A final direction of our research efforts lies in developing recombinant human hemoglobin systems for use as artificial blood substitutes and novel drug delivery vehicles. Having an alternative blood source which is guaranteed free of viral contamination is of obvious relevance. To provide the genetic handle for studies of reversible oxygen binding, cooperativity, plasma lifetime, molecular dynamics, electronic structure, and oxidation mechanisms, we have formed a multidisciplinary program and industrial partnerships to realize this important therapeutic goal.
Key Words Recombinant DNA, Novel Bacterial Transformation Systems, Gene
Expression Systems, DNA-Protein Interactions, Genome Organization/RFLP Mapping,
Monoclonal Antibodies, Bioengineering, Image Analysis, Diagnostic Reagents,
Pharmaceuticals, Fermentation, Biomass Conversion, Microbial Physiology, Biodegradation,
Site-Directed Mutagenesis, Protein Purification and Characterization, Protein
Processing and Secretion, Biocatalytic Conversions, Active Sites and Receptors,
Molecular Modeling, Biosensor Technology, Structural Biology, NMR
Current Research Funding
NIH, NSF, Corporate