Sligar Lab	Stephen G. Sligar I.C. Gunsalus Professor Dept of Biochemistry Dept of Chemistry College of Medicine Center for Biophysics and Computational Biology Director, School of Molecular and Cellular Biology

Research in the Sligar Laboratory

The cytochrome P-450 dependent mixed function oxidases play central and crucial roles in mammalian, plant, insect, viral, and microbial metabolism. Central problems being explored relate to the precise bioorganic and bioinorganic chemistry of dioxygen and substrate activation, the identity of metal-oxygen-carbon intermediates, and the structural and functional details of the catalytic event which control recognition and catalysis. Methodologies used include x-ray structure determination, cryoradioloysis and EPR, NMR, ENDOR Mossbauer, optical, infrared and EXAFS spectroscopies.

Additional NIH funded research focuses on molecular recognition questions, particularly in regard to small molecule, macromolecule and membrane recognition in metalloprotein mechanisms. Major recent efforts focus on the self-assembled Nanodisc system. Central questions relate to the mechanisms of self-assembly, the processes that control integral membrane protein incorporation, control of oliomerization state, fundamental investigations of substrate recognition in the cytochrome P450 monoxygenases, and the mechanisms of G-Protein Coupled Receptor signaling. Human cytochrome P450s involved in drug metabolism and steroid hormone biosynthesis provide another important research theme. We use the Nanodisc technology to aid in isolation, stabilization and functional understanding of drug-drug interactions and regulation and estrogen biosynthesis.

Another important research direction for our group is the development and execution of methodologies for the determination of the structure and function of biological assemblies - typically in the 5 nm - 500 nm 'mesoscale' size range. An ultimate goal is to be able to control the patterning of functional biological macromolecules at the nanometer scale and develop sensing modalities for single-molecule high throughput detection of macromolecular and small molecule recognition motifs. Toward this end we use the latest advances in atomic probe imaging, including scanning tunneling (STM), atomic force (AFM) and optical microscopies together with dip pen lithography (DPN), nanosphere lithography (NSL) and microfluidic sorting. This work is in collaboration with the Northwestern University Nanoscale Science and Engineering Center (NSEC). An immediate effort is the interfacing of our Nanodisc system for investigating integral membrane protein receptors and enzymes with the soft lithography, patterning and optical detection modalities of the NSEC.

The mechanisms by which living cells control migration is of paramount relevance to human health, including the metastasis of human carcinomas and all developmental processes. We collaborate with the Cell Migration Consortium, a large NIH GLU program headed by Alan Horowitz at the University of Virginia, which provides important live cell methodologies to our efforts. Our particular focus is to investigate cell migration and motility using de novo protein engineering to form in vivo reporters. In one project we have engineered a molecular force sensor in the form of an anti-parallel coiled-coil, calibrated this in collaboration with Dr. Ha in the Physics department using single particle FRET, inserted this into the filamin A of living cells and executed whole cell imaging. A similar approach is being used in collaboration with Dr. Chira in the Department of Cell and Structural biology in studies of the extracellular matrix of Drosophilia. A second effort also uses de novo design to generate novel genetically encoded reagents for chromoohore assisted laser inactivation (CALI) to define cellular functions and assembly organization. The final effort uses our Nanodisc methods to precisely quantitate the interaction of human integrins with scaffold proteins in the formation of focal adhesions and efforts to understand inside-out and outside-in signaling in integrin complexes.

Last updated: 7/12/08